Current status of flow cytometry in cell and molecular biology

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

Current Status of Flow Cytometry in Cell and Molecular Biology Guenther Boeck Institute for General and Experimental Pathology, University Innsbruck, Medical School, A-6020 Innsbruck, Austria

This review summarizes recent developments in flow cytometry (FC). It gives an overview of techniques currently available, in terms of apparatus and sample handling, a guide to evaluating applications, an overview of dyes and staining methods, an introduction to internet resources, and a broad listing of classic references and reviews in various fields of interest, as well as some recent interesting articles. KEY WORDS: Flow Cytometry, Cell sorting, FACS, Cell analysis, Fluorescence.  C

2001 Academic Press.

I. Introduction Flow cytometry (FC) is a method of measuring various physical and chemical characteristics of single cells or particles in suspension. It combines many of the advantages of microscopy, biochemical analysis, and computer development. Since the development of simple counting machines in the 1960s, FC has developed into an important analytical tool in almost all biological research fields—especially cell biology, immunology, and genetics—as well as in clinical research and routine laboratory testing. The principle of FC is similar to that of fluorescence microscopy, but in the case of FC, cells or other biological particles are measured one by one as they pass through a light beam. Over the past 20 years, improvements in data-handling techniques (software and hardware), dye synthesis, and staining techniques have improved the reproducibility of cell/particle handling and staining. Numerous specific fluorescent probes and staining procedures have been established, many of which preserve cell viability and therefore enable labeling and study of a International Review of Cytology, Vol. 204 0074-7696/01 $35.00

239

C 2001 by Academic Press. Copyright  All rights of reproduction in any form reserved.

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

240

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

variety of intracellular components of living cells. FC is used not only as an analytical tool but also for the powerful purpose of separating cells without loss of viability. In this article I do not attempt to provide an overview of FC during the past two decades. Rather, my aim is to focus on the current status of the field, concentrating mainly on recent applications and advances in cell and molecular biology. In so doing, I hope to provide a comprehensive first overview for newcomers to this exciting field, at the same time providing old-timers with a useful update on new materials and applications. It is not my intent to review recent advances in the clinical applications of FC, since many excellent reviews already exist on this subject (Keren, 1989; Vielh, 1991; Laerum and Bjerknes, 1992; Bauer et al., 1993b; Bogh and Duling, 1993; Macey, 1994; Schlossman et al., 1994; Orfao et al., 1999; Darzynkiewicz et al., 1999).

A. Relevance The basis of FC is the quantitative measurement of single biological particles on the basis of two or more parameters, usually size and fluorescence. The word particles is used in this review in its broadest sense, referring not only to various types of cells (including eukaryotic and hybrid cells), but also to nuclei, chromosomes, bacteria, cell organelles, liposomes, viruses, listed in decreasing order of size following Shapiro’s classification (Shapiro, 1995). Because most biological preparations are heterogeneous, single cell analysis offers many advantages. Even rare subpopulations can be analyzed, since FC permits screening of large numbers of particles within reasonable timeframes. Examples of FC applications are given in Table I. In addition to analysis, FC permits isolation of cells without loss of viability or particle purification without loss of structure. This is a very powerful tool. Practically anything that can be analyzed can be used as a criterion for sorting. Recent applications include use of cell sorting as a cloning device and subsequent PCR for single cell molecular characterization, sorting of human chromosomes, the isolation and subsequent propagation of hematopoietic stem cells for therapeutic use, and the purification of cell fragments and subsequent biochemical analysis.

B. The Roots: Prehistoric, Historic, Current There has been considerable synergy between the development of precise tools such as monoclonal antibodies, the development of various staining methods, and development of the analytical equipment.

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

Textures 2.0

241

TABLE I Some Examples of FC Applications Immunology Phenotypic analysis, developmental studies All that antibodies can detect Biochemistry Total DNA content/ploidity Cell cycle analysis Apoptosis detection Enzyme activities Functional studies Live-dead analysis Phagocytosis pH inside the cell Ca2+ flux detection Membrane potential Microviscosity of cell membranes Cancer research Residual analysis Molecular biology Chromosome analysis and sorting Rare event detection with PCR expanding Marker genes, green fluorescence protein

1. Antibodies as an Analytical Tool The development of antibodies as a highly specific tool provided a breakthrough in cell biology over the last century. Today an enormous variety of very specific monoclonal or pure polyclonal antibodies are used in numerous fields of research; for example, immunology for detection of antigenic structures, biochemistry for detection of proteins, and molecular biology for detection of peptide or specific DNA sequences. Some milestones along the way included the first side-chain theory of antibody formation (Ehrlich, 1897), detection of antitissue antibodies (Landsteiner, 1899), the theory of immunological tolerance (Billingham et al., 1953), and the development of hybridomas for mass production of antibodies (Koehler and Milstein, 1975). 2. Dyes and Staining Techniques In the latter half of the nineteenth century, the chemical industry focused on developing new dyes for the textile industry. But around 1900, Paul Ehrlich, experimenting with some of these dyes on biological materials such as tissues and blood cells, distinguished what is today known as acidophilic, eosinophilic,

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

242

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

basophilic, and neutrophilic granular leucocytes (Ehrlich and Lazarus 1900). Feulgen (Feulgen and Rossenbeck 1924) was soon able to stain DNA. Caspersson demonstrated the double amount of DNA in mitosis (Caspersson and Schultz 1938) and later developed a technique for making microspectrometric measurements on unstained cells (Caspersson 1950). Papanicolaou and Traut (1941) first showed the relevance of nuclear chemistry in the clinic. The need for new staining techniques was leading to the development of fluorescence measurements. For example, Friedman (1950) described the increased fluorescence intensities of malignant cells stained with a mixture of various dyes. This in turn led to development of a quantitative microphotometer for cancer diagnosis. Coons was able to couple antibodies with a dye (fluoroscein isothiocyanate, FITC; Coons, Creech, Jones 1941), and applied the technique to detect antigens with the FITC molecule in tissues (Coons and Kaplan 1950), a method that gained widespread use over the next few decades. The rapid developments taking place in the field at that time are illustrated by the increasing number of papers and reviews published (e.g., Miner and Kopac, 1956; Mellors, 1958). Today, several thousand dyes are available, not only for staining antibodies and detecting antigenic structure in multiple color experiments, but also for demonstrating functional properties of cells in combination with biochemical analysis. 3. Apparatus, Photometry, Data Handling Early analytic methods included microscopy (especially fluorescence microscopy), microphotometry, and image analysis systems. These were successfully applied in the study of tumor biology (Mellors and Silver, 1951; Mellors et al., 1952) and leucocyte differentiation (Kosenow 1952). However, with the introduction of DNA and RNA staining methods and the development of fluorescence-labeled antibodies, interest in automation and quantification of fluorescence intensity increased. Coulter’s method of cell counting (1956) was rapidly being adopted worldwide in clinical and research laboratories (Brecher et al., 1956, Mattern et al., 1957). Data-handling devices and computers brought statistics to cytometry. Quantitative fluorescence developed. The first home-brewed flow systems, technically little different from a fluorescence microscope, appeared in the 1960s. Kamentsky et al. analyzed cell cycles by measuring DNA content (1965); Sweet investigated cell depletion (1965), providing the basis for Fulwylers work on cell separation (1965); blood granulocytes and lymphocytes were purified for the first time by Van Dilla et al. (1967). Other papers published in the 1960s provide insight into the rapid technical progress as FC was applied in various biological fields (Koenig et al., 1968; Hulett et al. 1969; Saunders and Hulett, 1969; Van Dilla et al., 1969; Kamentsky and Melamed, 1969; Finkel et al. 1970). The early 1970s brought the first commercially available cell analyzers: the Phywe, basically a DNA measuring machine around a microscope;

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

243

the Cytofluorograf, a machine equipped with a laser that could discriminate between living and dead cells and perform DNA measurements; and the HemalogD (Technicon), a machine for analyzing leukocytes. With the introduction of lasers as strong and stable excitation light sources, multicolor staining and detection of weak immunological staining became possible. Herzenberg’s group used an argon (Ar) ion laser to analyze and sort antibodylabeled cells (Bonner et al. 1972). Becton/Dickinson started their production with similar machines. Over the next two decades a number of important advances were made. Steinkamp et al. (1973) designed a multiparameter system. Gray (1975) started work with chromosome measurement and sorting. Horan reviewed advances in quantitation (1977), Hercher et al. (1979) described detection of single viruses with less than 100 fluorescein molecules, and Nguyen et al. (1987) described detection of single molecules. Hand in hand with the development of optical devices, data-handling systems were refined such that nontechnical personnel were able to operate benchtop cytometers. Today a variety of machines are available, ranging from small and “easyto-use” machines to big (and expensive) ones. Lower-end machines at a minimum can analyze cells in double color staining and provide information on various morphological parameters.

II. How It Works Various systems exist for getting cells to pass through a light beam. General references and detailed technical applications can be found elsewhere (Gray, 1989; Melamed et al., 1990; Shapiro, 1995; Van Dilla, 1985; Watson, 1992; Robinson, 1993; Tanke and van der Keur, 1993; Darzynkiewicz et al., 1994; Omerod, 1994). This review provides only a short introduction to these concepts.

A. Generalized View The principle of a flow cytometer can be explained as follows. A flow cytometer consists of three main parts: a flow system, a light source together with some lightcollecting optics, and a data analysis system. In almost all flow cytometers, the light source is a laser, selected according to the excitation wavelength(s) required, and the data analysis system is a computer with appropriate software. The flow system serves to channel cells from a sample tube to an intercept point with a focused light beam. A cell suspension is surrounded by a buffer “sheath fluid,” usually physiological saline, which not only prevents clogging but also serves to align the cells (hydrodynamic focusing). The pressure of the cell suspension and the buffer sheath (preferably applied with pressurized air) is adjusted in such a way

10/31/2000

05:27 PM

244

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

that one cell after another crosses the light beam. Two systems exist: the closed system and the stream-in-air system. Closed systems, in which the cell suspension is injected in a small flow chamber, are used mainly for analyzers. Closed systems have good optical quality and therefore require only small lasers. Stream-in-air systems are used by almost all cell sorters. They have a nozzle with an orifice through which one cell after another can emerge, falling immediately through a focused light beam. The optical quality of these systems is poor because a lot of light is lost; they therefore require lasers with much greater output power. In both systems, light signals are captured from individual particles by an array of light detectors that surround the measuring point, together with appropriate lenses, beam splitters, and optical filters. Particles passing through the strong light field scatter the laser beam light in all directions. The intensity of scatter in the forward direction (forward scatter, FSC) is approximately proportional to the particle size and is not much affected by its shape or surface properties. However, scatter at right angles relative to the incident laser light (90 degree scatter or side scatter, SSC) is proportional to the quantity of granular structures in and surface roughness of the particle. Two lenses collect signals: one located in front of the laser, one at 90 degrees to it. Scatter signals are of complex nature, due to diffraction, reflection, and refraction of light from the particle. For this reason, information on size and structure of a cell from scatter signals is only true for “normal” cells. For big particles (>30 m, e.g., cell aggregates) and for very small particles (<1 m, e.g., endosomes), scatter signal intensities require special interpretation. As a general rule, what is seen in a cytometer should be in agreement what is seen in a microscope. Multiangle scatter detection systems, which were introduced by Salzman et al. (1975), are seldom used routinely because of the difficult interpretation of the results (Price et al., 1978, or Schafer et al., 1979) and the nearly impossible verification by microscope. Light scatter signals probably prove most valuable not for providing absolute information on cell size but for monitoring changes in cell size—for example, during development in cell culture—and for comparing cell types. Also, functional parameters such as apoptosis, live-dead discrimination, or degranulation of cells can be detected by light scatter signals (Darzynkiewicz et al., 1992). Besides the measurement of cell size and complexity, fluorescence can be detected at several wavelengths. Cell components or functions can be specifically stained with dyes (nucleic acids, proteins, specific receptors, intracellular ion molecules, etc.). These complexes can absorb the incident laser light and emit fluorescent light, due to their excitation and emission spectra. Fluorescence emission is about 10,000-fold weaker than scatter light. In an ideal situation, the fluorescence signal is proportional to the number of dye molecules present. Fluorescent light is fractionated by a series of optics, beam splitters, and optical filters into parts of the optical spectrum, thus allowing multiple color detection simultaneously and independently. The signals are condensed to data by photomultiplier tubes (PMT)

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

245

and amplifiers (logarithmic or linear), and displayed and stored on a computer equipped with appropriate software. With such a FC (a typical example is the FACScan from Becton/Dickinson), cells can be analyzed according to morphological structures (FSC, SSC) and fluorescence intensities (in the three fractions of the optical spectra: green, yellow, red).

B. Advanced Systems Many people have built their own cytometers, and many others have adapted existing machines for their specific needs. The following sections are restricted to discussion of commercially available models, however. 1. Additional Lasers/Colors Standard analyzers use a blue laser as excitation light source. In addition to morphological information, this laser can provide up to three fluorescence channels. More advanced analyzers may have two or three lasers of different wavelength (usually one UV, one blue, and one red) and can excite up to eight different dyes (Roederer et al., 1997). 2. Sorting Various techniques exist to separate particles. The most common is the streamin-air system, in which the continuous stream of sheath fluid emerging from the nozzle tip is separated into well-defined single droplets (each containing a single particle) by mechanical vibration of the nozzle. Droplets containing particles to be separated are charged with various electrical intensities, separated from other droplets by passage through a strong electric field, and collected in separate tubes. Up to about 3,000 cells can be sorted per second without loss of cell viability. Other sorting systems, such as the fluidic switching system (Duhnen et al., 1983), have the advantage of being closed systems, well suited for handling biohazardous samples, simple (i.e., without necessary adjustments of streambreaking point, etc.), and capable of handling large objects (Gray et al., 1989), but they have the great disadvantage of being slow. 3. Enhanced Sorting Capabilities With care, it is possible to increase system pressure or use syringe systems for sample injection such that sorting and analyzing rates can exceed 10,000 cells/sec (Peters et al., 1985). This is important when sorting low percentage subpopulations in order to get sufficient material for further biochemical analysis (see Section III.B.5.b. Chromosomes and PCR) or for enrichment of rare particles (see also

10/31/2000

05:27 PM

Cytology

PS012-05.tex

246

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

Section II.E.3. Limits), among them human fetal cells (Leary et al., 1994) or hematopoietic stem cells (Sasaki et al., 1995). Four-fold sorting devices exist that can separate up to four populations instead of the usual two “positive” and “negative” populations (Arndt-Jovin and Jovin, 1974). Automatic “cloning devices” are available as an option on some commercial cytometers. These permit sorting of one and only one cell with known characteristics into a tube or microtiter well for single-cell PCR or further expansion. Huge numbers of plates can be filled with such an automated device in a very short time. 4. Time Resolution As particles travel through a focused laser beam, the detected signals increase upon entrance into the beam, reach a peak (“height”) at the center, and decrease as they exit the beam. In everyday flow cytometry, the peak height provides a measure of the light intensity. With an option called a pulse processor, the pulse width (which is the time of passage of the particle through the beam) and pulse area (which is an integral of all light from the particle) also can be processed. Such information is essential, for example, in DNA-analysis (Monroe et al., 1982), for distinguishing between single cells and doublets (Sharpless et al., 1975; Sharpless and Melamed, 1976). For some analyses, further mathematical calculation, such as subtracting one signal from another or dividing two signals to get a ratio, provides useful information. Such calculations can be done online with an electronic device, or retroactively by computer after the data have been collected. For a small laser-spot (<1 m), particle size information can be obtained using slitscan methods, which involve collecting signals of height, area, and width at certain time intervals (Wheeless and Patten, 1984; Sharpless et al., 1977; Cambier et al., 1979; Leary et al., 1979; Peeters et al., 1989; Dubelaar et al., 1989; Rens et al., 1994). An approach different from the time-of-flight sizing system uses time-resolved information about the dye itself (Pinsky et al., 1993). This phase-sensitive detection method is based on the following premise. Laser light is modulated with high frequencies; fluorescence emitted is also modulated, but with a small delay due to the lifetime of the excited state of the dye molecule. Different dyes have different delays (Steinkamp and Crissman, 1993), as well as autofluorescence, which can be separated with phase shift amplifiers. Such instruments have an excellent signal-to-noise ratio, especially when long lifetime substances such as chelates (Condrau et al., 1994a; Condrau et al., 1994b) are used. The disadvantage of such systems is the long measuring time required.

C. Simpler Systems Some simpler machines are available that generally have fewer buttons, are strictly software driven, have no sorting option, and the excitation light source is mostly

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

247

an arc-lamp (which is not too bad for some dyes, as shown by Peters 1979). These less expensive machines are built for very rapid measurements in laboratory use, but are also used in underdeveloped countries D. Data Display and Data Analysis Light signals collected by the optics and converted to electric pulses can be processed to data, displayed, and stored in various ways: the method of choice is the list mode storage, in which measurements from each individual cell are stored separately. The type of display chosen depends on the aim of the experiment as well as on the software capabilities, but some guidelines should be taken into account. 1. The Single Parameter Display (Histogram) In this display the distribution of one parameter—for example, green fluorescence intensity—can be displayed versus another such as cell number, number of events, or percentage of cells. Figure 1a uses antigen expression on mouse thymocytes as an example. Note that in immunofluorescence work, fluorescence intensity is almost always plotted on a logarithmic scale; on the scale used in Fig. 1a, a cell on the far right end of the scale would be 10,000 times brighter than that of one on the far left end. In contrast, cell cycle analysis is almost always plotted on a linear scale, so doubling the distance from the left end (the zero point of the x-axis) would mean a doubling of the fluorescence intensity.

FIG. 1a Single parameter display (histogram): Green fluorescence intensity (x-axis) of 40,000 cells plotted on a logarithmic scale; curve (Gaussian-like shape) statistics described by the mean/median value. Experimental system: immunofluorescence staining of mouse thymocytes (BALB-c strain, five weeks old), staining of the glucocorticoid (GR) receptor with an a-GR monoclonal antibody, detected with FITC-labeled second antibody.

10/31/2000

05:27 PM

248

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

In biology, most distributions more or less follow a Gaussian curve, which is characterized by two numbers, the mean and the standard deviation. In FC, the arithmetic mean or average fluorescence intensity, calculated statistically, is often used as a measure of the expression of an antigenic structure, or the function of a cell, etc. In an ideal Gaussian distribution, the arithmetic mean FI would be identical to the median FI, which is the intensity at which 50% of the cells are dimmer and 50% brighter. In reality, however, it is often preferable to use the median FI, as it is less affected by values that may be lost at the extreme ends of the scale. The standard deviation, expressed as CV (coefficient of variation), is important mainly when analyzing DNA preparations, as it provides an indication of the quality of the preparation. A narrow fluorescence peak (low CV) also is essential for cell cycle analysis (see later in Section III.B.3.b). When described by numbers in this way, Gaussian distributions can be easily compared. For example, expression of a certain antigen could be compared before and after mitogenic stimulation; doubling of the median value can be interpreted as doubling the surface antigenic expression (this will be discussed in Section II.D.4). The situation is more difficult when it comes to comparing two separate or overlapping distributions of FI. An example is given for human lymphocytes in Fig. 1b. In this example, the mean of all events would provide meaningless information, so it becomes necessary to calculate the mean FI of the “dull” or “unstained” cells separately from the mean FI of the “bright” or “stained” cells and give percentages in each distribution. When differences in FI between stained and unstained cells are smaller, the two distinct populations “melt” into one another with a more or less pronounced shoulder, and this type of gated analysis may not be possible. Discrimination between “stained” and “unstained” cells depends then on the

FIG. 1b Single parameter display (histogram): Fluorescence intensities of 10,000 cells, 54% negative (unstained, dull), 45% positive (stained, bright). Experimental system: immunofluorescence staining of human whole blood lymphocytes with an a-CD3 monoclonal antibody, labeled with FITC. Stained cells are T-cells.

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

249

background controls, the distribution of fluorescence staining, and the experimental controls (Chase and Hoffman 1998), as well as the computer software available. Almost all computer programs offer the option of subtracting one histogram from another. So the “unstained” control sample A can be subtracted from a mixed population-curve A + B, leaving an uncontaminated population B for which the mean FI can be calculated. 2. Dual Correlated Parameters Display As described previously, one of the main advantages of FC compared to related techniques is the ability to correlate two or more parameters. The display of such data can be performed in various ways, but preferably with a dot plot (Fig. 2a),

FIG. 2a Dual correlated parameter display (dot-plot): Distribution of 50.000 cells (mouse thymocytes, BALB-c strain, five weeks old) in an immunofluorescence double-staining experiment: x-axis yellow fluorescence of a-CD4-PE, y-axis red fluorescence of a-CD8-Cy. The display is divided into quadrants for analysis: UL, upper left, represents CD4 negative, CD8 positive cells (5%), UR, upper right, represents cells containing CD4 and CD8 epitopes (59%); LL, lower left, represents cells not stained by either antibody (23%), and LR, lower right, represents CD4 positive, CD8 negative cells (13%). The data can be simplified to the histograms shown on the right (for the a-CD8) and at the bottom (for a-CD4 staining), although information on correlation is lost with this type of data display.

10/31/2000

05:27 PM

Cytology

250

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

FIG. 2b Dual correlated parameter display (contour plot): Data identical to those in Fig. 2a.

where each dot represents data from one single cell. Quadrant statistics are used to gain useful information from these plots. In our example, mouse thymocytes were stained with two monoclonal antibodies (anti-CD4 and anti-CD8) in direct immunofluorescence tests. The anti-CD4 was labeled with the fluorescent dye PE (yellow fluorescence) and anti-CD8 was labeled with Cy (red fluorescence). From the dot plot, it is clear that the thymocyte population contains four separate subpopulations: one that stains brightly with both antibodies (bright yellow/bright red); one that stains with anti-CD4 only (bright yellow/dim red); one with antiCD8 only (dull yellow/bright red); and one not stained with either (dull yellow/dull red). The data can be condensed to two single histograms, as shown below and to the right of the dot-plot, though any information on correlation is then lost. Other ways of presenting the same data include the contour-plot (Fig. 2b) and the three-dimensional display (perspective plot, Fig. 2c), which usually is preferred in time-dependent experiments. 3. Setting Gates In any normal experiment, the population of living cells is almost always contaminated with some sort of debris, cell clumps, or unwanted subpopulations. Setting gates can help with obtaining data from a defined subpopulation. Various gates and logical combinations of gates can be set, depending on the software capabilities, and can be used to detect events covered by unwanted data (Fig. 3). 4. Quantification Because the scale of a histogram is relative, so are the mean values. This means it is only possible to compare results within a single experiment, when hardware

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

251

FIG. 2c Dual correlated parameter display (perspective plot): Data identical to those in Fig. 2a.

settings, such as the gain of amplifiers, are constant. Is it possible to calculate the number of binding sites or antigen structures per cell from FC data? This question is of particular importance for clinical applications, and it is the goal of many researchers to calibrate fluorescence intensity (as discussed in a special issue of Cytometry by Gratama et al., 1998; Serke et al., 1998; Schwartz et al., 1998; Henderson et al., 1998). Commercially available kits have been developed for this purpose. These consist of beads, each embedded with a known number of dye molecules (1,000 to 1,000,000) in an inert matrix. These kits are stable over several years and have low CVs. The FI of an unknown sample is compared with that of the beads (Fig. 4). Calibration curves can be calculated by using beads with different known MESF (molecules of equivalent soluble fluorochromes; Schwartz et al., 1993). Although calibration bead kits are available for most of the commonly used dyes, a few uncertainties remain concerning the validity of the results because (a) dyes can change their excitation/emission spectrum slightly with binding to an antibody or some target structure, (b) some dyes (FITC is a prominent example) change their FI significantly with pH, and (c) although a solution of beads with dye molecules on the outside may share the same environment as the cells to be measured, the configuration may be less stable and the dye molecules on the surface may bleach, i.e. loose fluorescence intensity due to decomposition of dye molecules. Care must therefore be taken when fluorescence intensities of biological samples are compared with those of a stable dye embedded in a bead. This is even more true with calibration of scatter signals to quantify particle diameter (as will be explained in the next section). Semi-quantitative methods for comparing of curves, mean values, etc., are much less complicated and have proved extremely consistent within each laboratory (Nicholson and Stetler-Stevenson, 1998). A few examples to be mentioned here are the successful quantitation of receptor-ligand binding

10/31/2000

05:27 PM

252

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

FIG. 3 Setting gates: Data identical to those in Fig. 2a. By setting gates onto CD4/CD8 staining, a third staining of the T-cell receptor (a-TCR−biotinylated with FITC streptavidine) can be demonstrated: CD4/CD8 double-positive stained cells together with double-negative cells expressed T-cell receptor at a lower level than CD4/CD8 single-positive cells.

(Bohn 1976, 1980; Chatelier and Ashcroft, 1987; Uckun et al., 1989; Krause et al., 1990). Flow cytometry offers one major advantage over conventional radioligand binding assays, namely the possible detection of subpopulations (Corsetti et al., 1991, 1993). Scatchard and Lineweaver-Burk plots, common in radioassays, were applied to flow cytometry assays for analysis of estrogen receptors on human carcinoma cell lines (Benz et al., 1985) for analysis of high-density lipoprotein

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

253

FIG. 4 Quantification with beads: Same data as seen in Fig. 2b, overlaid by data of a mixture of six various bead suspensions containing different MESFs (molecules of equivalent soluble fluorochromes). Each of these standard beads emits as much fluorescence as (from left to right): “zero,” 20.000, 50.000, 100.000, 300.000, 750.000 molecules of FITC in solution, thus enabling plotting of a standard curve.

(Traill et al., 1987a) and low density lipoprotein (Traill et al., 1987b) receptors on subpopulations of lymphocytes. 5. Data Presentation Consensus The International Society for Analytical Cytology (ISAC) has established guidelines aimed at enhancing the reproducibility of results and facilitating comparison of data from different laboratories. The guidelines recommend that authors provide detailed information on the following in each manuscript: (a) aquisition—the instrument used, together with settings, compensation for multicolor measurements, software, excitation light and power, filter, and fluorophores; (b) data display— axes labels, scales, number of events, markers, and gates.

E. Limits Methods exist to handle dimly fluorescent particles (Chase and Hoffman, 1998) or to detect single molecules as described earlier. In most experiments, the limiting factor for sensitivity seems to be autofluorescence. Most mammalian cells have autofluorescence equivalent to about 1,000 fluorescein molecules per cell. This is primarily due to the presence of pyridine and flavin nucleotides that are excited with the UV light and blue light (even more than UV) used in daily cytometry (Benson et al., 1979). In cell line cultures, autofluorescence increases to such an extent that it can even be used as a parameter of growth and development (Aubin, 1979; Jongkind et al., 1982; van de Winkel et al., 1982; Liang and Petty, 1992). Obviously,

10/31/2000

05:27 PM

Cytology

PS012-05.tex

254

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

it is not possible to detect expression of an antigenic structure expressed in as few as 100 copies in the cytoplasma of such a cell. In some experiments, mathematical subtractions have been used to lower background (Corsetti et al., 1988). Alternatively, the problem can be solved by use of longer wavelengths for excitation; none of the autofluorescence-causing substances are excitable with red light. Use of red-excitable dyes in immunofluorescence work and biochemical and functional studies, as well as the use of small, cheap, red laser diodes, automatically increases the sensitivity of flow cytometry due to better signal-to-noise ratio. As with time-resolved cytometers, sensitivity also can be increased with low throughput and photon-counting systems (Agronskaia et al., 1998). Analysis of rare events is another problem that is of particular interest in cancer work (Gross et al., 1995; Atwater and Corash, 1996), for fetal cell detection (Lewis et al., 1996), and for cloning experiments. Analysis with high flow rates (more than 104 events/sec) is not complicated from a technical point of view (Rehse et al., 1995). The problem lies in identifying stained cells that represent less than 0.1% of the cell population on a dot plot or histogram. The ability to do so depends mainly on the variance of fluorescence intensity distribution: the brighter the stained cells are, the better they are separated from an unstained population and the more readily they can be detected. Staining of unwanted cells with a different dye (antibody) or staining subpopulations with more than one stain (cocktail staining) helps improve the statistical reliability (Gross et al., 1993; Rosenblatt et al., 1997). For really rare cells (less than 1:107), the limiting factor seems to be the huge number of cells (and the time needed to analyze all those cells). Assuming a 1:107 proportion and needing a few hundred events, analyzing times of hours or days may be required, even with high speed cytometers. Sorting of those rare events necessitates special attention, as mentioned in Section II.B.3; generally, the purity of the sorted fraction decreases with increased sorting rate.

F. Sorting Considerations Sorting can be done at an acceptable rate (>2,000 events/sec) only with a “streamin-air” system. Rate of sorting can be increased by simply increasing air pressure. But upon reaching a certain pressure, dissolved air in the sheath system tends to decompress very rapidly after emerging from the nozzle tip. While this does not much affect stable particles such as chromosomes or nuclei, it can be a problem for living cells. Syringe systems were therefore developed together with buffer sheath degassing systems (Peters et al., 1985). Speed limit is then reached with dead time of the data processing system or with the droplet formation of the fluidic system. The purity of the sorted fraction will never be 100% (but almost). Although flow cytometry sorting is a good method for purification without loss of material, it can be slow when sorting rare events or getting a sufficient amount of material for further biochemical analysis. It is a good strategy to enrich the wanted events

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

255

with some conventional methods, among them magnetic bead preselection, surface adherence, density centrifugation, or even raw flow cytometric sorting with high speed, disregarded purity. In summary, the limitations of sorting have been extended such that single cells can be separated from millions of others, sterile and without loss of viability, so they can be expanded, cultured, and cloned, or material can be collected for subsequent PCR.

G. Standards and Quality Control Besides the question of quantification, standards are used in FC in several ways: first and foremost to test the performance of the cytometer. As mentioned earlier, beads with different amounts of embedded fluorophor molecules are available commercially. Almost all of these bead kits have a very small variance of fluorescence, e.g., <2%, so routine, daily running of these beads should give a reproducible fluorescence histogram with a variation of less than 2%, unless the nozzle is clogged, laser instable, etc. To test the quality of an experiment, especially a DNA experiment, where low CVs are required, known test standards with low fluorescence variance may be added to the test sample. Nuclei from chicken erythrocyes (DNA content of 35% of the human diploid value) and rainbow trout erythrocytes (80% of the human diploid value) should both give small variance of staining under proper test conditions (more details in Section III.B.3.b). For DNA content of plants, Johnston suggested similar standards (Johnston et al., 1999). For immunofluorescence staining, variations in FI are in the range of 10%, mainly due to experimental variations (Vesely et al., 1996). Using beads, a dayto-day variation of the test system is detected rather than an absolute number of antigenic molecules (due to the problems mentioned earlier). Using biological standards, CD4 antigen is expressed with a CV of 5% among healthy blood donors and estimated to be about 50,000 antibody binding sites per cell (Hultin et al., 1998). Sometimes an absolute number of events per volume (for example, in hematology) is of interest. Counting cells per time interval, as is done in a coulter counter, is an inaccurate method in FC. Some people have good experience with mixing their experimental sample with a known number of beads, or similar stable particles with completely different characteristics (to avoid interference with the sample) to compare both populations and counting absolute numbers (for example in human work: Matzdorff et al., 1998, in chicken: Burgess and Davison, 1999). Beside these efforts for quantification and calibration in FC, a substantial claim in standardization and quality control of immunofluorescence test exists. Guidelines for immunophenotyping, clinical practices and methods can be found elsewhere (Landay and Muirhead, 1989; Parker et al., 1990; Mayall, 1993; Nicholson, 1994; McCoy and Keren, 1999).

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

256

Textures 2.0

GUENTHER BOECK

III. Measurable Parameters A parameter describes a physical or chemical characteristic of a particle either measured directly or with an associated reagent molecule, the reporter probe, which in FC is a dye molecule. According to Shapiro’s classification (Shapiro, 1995), parameters are classified either as intrinsic structural or intrinsic functional (when structures or functions of cells are measured without any additives) or as extrinsic structural or extrinsic functional (when dye molecules are used to stain structures or functions). This classification is not strictly unique, but overlapping. Dye molecules used to measure extrinsic parameters need special attention (the requirements for the dye will be discussed later), and many competent chemists are doing a good job of finding and synthesizing new compounds for various staining methods.

A. Just Native: What Can Be Measured without Staining (“Intrinsic”)? Besides merely counting particles, information collected from native preparations comes mainly from light scatter signals. 1. Light Scatter Light scatter signals are used in all FC experiments. The forward scatter (FSC) signal is used to set the trigger that tells the flow cytometer that a particle is there and information should be processed. Everything “smaller” than a given threshold is estimated to be debris, and its data is normally discarded. As mentioned earlier, scatter signals are of complex nature, but with some restrictions they carry useful information about size and structure (McGann et al., 1988) and can be used for analysis of unstained subpopulations (Salzman et al., 1975; Kerker et al., 1979; Loken et al., 1990; Rabinowitz et al., 1992; Ost et al., 1998). With “normal” cells, the small angle forward scatter signal is more or less proportional to the particle volume, but is strongly affected by technical details, such as lens aperture, laser wavelength, and refractive index of the cell membrane, etc. (Scherer et al., 1999). Scatter signals in a series of other directions (multiangle scattering) can be collected with some machines with complex results but have been used to identify subpopulations of algae (Price et al., 1978) and human fibroblasts (Schafer et al., 1979). FSC and side Scatter (SSC) signals have been applied in various fields (Ulicny, 1992), including cancer research (Mohler et al., 1987 or Duque, 1993), characterization of nuclear division (Giaretti and Nusse, 1994), and microbiology (Fouchet et al., 1993). Dead cells generally have reduced FSC mainly because of the variation of their refractive index (Loken and Herzenberg, 1975; Melamed et al., 1990).

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

Textures 2.0

257

For very small particles, scatter signals seem to fulfill Rayleigh criteria, meaning that FSC and SSC are strongly dependent on particle size, and are not affected by internal structures (Hercher et al., 1979; Zarrin et al., 1987; Mueller et al., 1994). For large cells, the FSC signal increases in inverse proportion to volume, due to particle light absorption and absorption of the first lobe of scatter signals by the obscuration bar. SSC signals, normally used in haematology to separate lymphocytes from monocytes and granulocytes, also can be used to follow vacuoles (Dubelaar et al., 1987) or changes following staining: lymphocytes labeled with antibodies conjugated to colloidal 40 nm gold particles increase SSC without affecting antibody staining (Boehmer and King, 1984; Dubelaar et al., 1987). 2. Fluorescence As mentioned earlier, autofluorescence is mostly due to the presence of pyridine and flavin nucleotides, which are excited with UV-blue light. It is the limiting factor in sensitivity for blue excitation, because although it can be reduced (by propriety narrow band filters), it cannot be eliminated. However, autofluorescence can provide useful information concerning subpopulations (Havenith et al., 1993) or the metabolic state of a cell, mainly the redox state (Thorell, 1980). In hematology, neutrophils and eosinophils differ in autofluorescence (Weil and Chused, 1981). Another source of autofluorescence is the accumulation of pigments: lipofuscins for example, accumulate in elderly mammalian cells (Jongkind et al., 1982; Jongkind and Verkerk, 1984). Chlorophyll and phycobiliproteins are present in a variety of algae and bacteria, and the fluorescence can be used to classify phytoplankton populations (van den Engh et al., 1985; Demers, 1991; Lloyd, 1993).

B. Reporter Molecules, and What They Can Tell Us (“Extrinsic”) Structure (or function) can be stained with dyes by various mechanisms: 1. Molecules can be bound covalently to a structure. This is done mainly in immunofluorescence work, where dye molecules are bound to antibodies. 2. Molecules can have a high affinity to a cell structure, so an enrichment of molecules in a certain compartment can be obtained (dyes with high lipid solubility can stain cell membranes; dye molecules outside membrane only increase unwanted background fluorescence). 3. Quantum efficiency of dye molecules is increased with binding to a structure. This is done in DNA staining where only bound molecules emit significantly fluorescence light. 4. In response to binding, dye molecules can change their excitation/emission spectra. An example is the Ca2+ staining dye Indo-1. Excited with UV light,

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

258

Textures 2.0

GUENTHER BOECK

the emission maximum is mainly in the blue region, with binding to Ca2+ ions the maximum is shifted to shorter wavelength. 1. General Requirement for a Dye A dye must obviously fulfill certain requirements for successful staining, the first of which are physical requirements: 1. The wavelength of a commonly used laser (Ar-laser, HeNe-laser, etc) must correspond to the absorption spectrum (ideally the maximum) with a high absorption rate. 2. The emission spectrum should be narrow to avoid overlapping of signals from various dyes. With multicolor staining, it should be possible to completely separate (with no overlap) the emission spectrum of each dye using optical filters. This turns out to be a very stringent requirement due to the small range of the visible spectrum of only one octave (coarse: 400–800 nm). Because absorption and emission spectra are for physical reasons more or less mirror-images, the requirement of one laser excitation and multicolor emission without overlapping is hardly feasible. To separate the fluorescence emission of various overlapping dyes, hardware components such as differential amplifiers or software subtraction are used. 3. The quantum efficiency, which is the probability of an absorbed photon being transferred to an emitted photon, should be high. The second set of requirements is biological, among them: 1. The specificity, which is the ability of dye molecules to stain wanted structure without evoking nonspecific or background staining. 2. The stain should neither be toxic nor cancerogen (at least for experiments with living cells). For immunofluorescence studies, the dye should not be an immunogen, disturb the immunologic reaction, or stimulate the cells in any way. These requirements all depend, of course, on the experiment. Dyes under consideration should be checked carefully in this respect. It should be emphasized here that binding of some dyes is stable, some reversible (depending on environmental conditions such as pH), and some in equilibrium with the surrounding (can be washed out). This should be taken into consideration especially for longer lasting experiments. In most cases, these requirements are only partially fulfilled by common dyes, all of which have advantages and disadvantages. Years ago, reproducible staining was more or less a thing of luck and probably the phrase “in our hand it does not work” arose at that time (Horobin, 1969; Scott, 1972). Although the situation is improving, and new and more specific dyes are becoming available, it is still a good strategy not to place too much trust in the suppliers: each dye should be assessed in the specific experimental situation for which it is

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

259

to be used, along with every experimental control imaginable. In additional, it is essential to check results with a simple fluorescence microscope. Flow cytometric data and fluorescence microscope images should always agree. 2. Fixation and Permeabilization Obviously dye molecules must reach their binding sites, which is not a problem for cell surface structures (membrane stain or immunotyping). For intracellular staining, various methods exist for getting dye molecules through cell membranes. The most simple situation is to use uncharged and/or lipid soluble molecules, which can cross intact cell membranes (for example the DNA-staining dye Hoechst33342). The situation is similar for diacetates and acetoxymethyl esters (AM). Both substances are able to cross cell membranes, cleaving off the acetate/acetoxymethyl ester group with esterase, an enzyme present in almost all cells, thereby releasing the fluorescent dye, which cannot cross the cell membrane in this configuration. The straightforward way of making intracellular structures accessible is to make some holes in the cell membrane, e.g. by electroporation, where electrical pulses damage cell membranes. Dyes (and other substances) can enter the cell (some other substances may exit the cell) until the membrane is built up again. Some cells can survive this procedure. However, the most common method is to fix cells and permeabilize cell membrane with chemical agents, such as acetone, ethanol, methanol (coagulant), or formaldehyde (non-coagulant) in various combinations and sometimes in combination with detergents, such as saponin, Tween, Triton X-100, and digitonin (van Ewijk et al., 1984; Hopwood, 1985; JacobbergerFogleman and Lehman, 1986). Fixed cells are dead, which is essential for working with biohazardous material, and more or less stabilized. Stabilized cells can be stored from a few days to a few weeks with special storage buffers (Ormerod, 2000), which provides a great advantage for busy flow cytometry centers or when technical difficulties are encountered in the unit. Beside these advantages one must keep in mind that fixation, permeabilization, and staining are somehow diametrically opposed to one another. The cell membrane should be permeabilized, but all structures should be conserved for detection by antibodies, and additional washing steps should not remove stained structures. Sometimes immunofluorescence surface staining is performed before fixation. However, none of the methods is universal and each experimental setup needs special attention (Tiirikainen, 1995). A different approach is to use some kind of Trojan horse to gain entrance into living cells. Examples include liposomes (lipofection, common in molecular biology work to infect living cells with DNA constructs), and endosomes with surface bound (e.g., lipid soluble) dyes (Boeck et al., 1997). In addition, one must keep in mind that any staining obtained after cell permeabilization is not an exclusive reflection of intracellular staining, but rather a combination of intracellular and surface staining.

10/31/2000

05:27 PM

Cytology

PS012-05.tex

260

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

3. Structural Staining a. Antibodies An incredible variety of polyclonal as well as monoclonal antibodies is now available commercially. (Details on antibody production, purification, and theory can be found in many immunological publications.) A dye to be used for immunofluorescence staining must fulfill two requirements in addition to those mentioned above. It must not disturb the immunological function of the stained antibody (therefore, the molecule must not be linked to the variable part of the antibody molecule) and it must not exceed a certain size. As the detection limit for most “average” cytometers is limited to a couple of thousand dye molecules per cell (as discussed in Section II. E), each antibody should be labeled as heavily (brightly) as possible to maximize sensitivity in case of low antigen expression. It is now known that for not more than five small dye molecules per antibody, molecule fluorescence is optimal. With more dye molecules per antibody, fluorescence intensity decreases due to inner filter effects and/or concentrations quenching, and more heavily labeled antibodies are more likely to precipitate out of solution. Attempts to couple more molecules using some kind of spacer to increase fluorescence intensity have also failed (Hirschfeld 1976; Hercher et al., 1979). This leaves the technique of indirect staining as the method of choice for increasing the test sensitivity. Indirect staining, which is the detection of a bound antibody with a second step reagent (fluorescence labeled antibody) against the first one, has several advantages: (a) The immunological situation is more defined, the first antibody not being disturbed by dye molecules; (b) the second (labeled) antibody (“conjugate”) binds to the Fc-part of the first antibody, so normally more than one second antibody can be bound, and the sensitivity is increased by about five-fold; and (c) Good controls for nonspecific fluorescence can be performed by incubating the sample with the second antibody only. A drawback to indirect immunofluorescence is the possible cross-reactivity between antibodies of various species, especially with multicolor staining. This can partly be solved by using a heavy chain specific second antibody, and working with monovalent or bivalent Fab2 antigen binding fragments and/or some blocking steps during performance of the test. Another trick for signal amplification is the biotin-avidin system (Bayer and Wilchek, 1978). Various sandwich layers with biotinylated antibody together with strepavidin-dye can increase staining intensities many-fold (Zola et al., 1992, demonstrated this in cytokine detection). However, all these systems are reasonably good mainly for surface staining. To enter cytoplasm, regardless of how the cell membrane is crossed, small molecules were preferable to diffuse and stain all structures. Antibody molecules (150 kDa) are already quite big for this diffusion, but an antibody, for example labeled with PE, a commonly used dye in immunofluorescence of similar size as an antibody, will not successfully stain intracellular antigenic structures.

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

261

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

So for antibody staining within a cell, crossing the cell membrane and finding the structure will always be tricky. In addition, the number of antigenic structures is normally much lower inside the cell than on the cell surface, so staining will be weak, and amplifying with indirect staining is not recommended. A different approach for increasing sensitivity is to stain antigenic structures with particles (microspheres, beads, etc.). These particles consist of a primary inert surface and/or a matrix, the inside of which is filled with dye molecules and the outside is coated with antibodies, antigens, or prepared for avidin/biotin systems (Bhalgat et al., 1998). This method is sensitive, the dye molecules are very stable, but obviously the reaction is not stoichiometric, and problems can occur with clumping and/or unspecific staining. Another problem is that monoclonal antibodies that are supposed to be the same in fact come from different clones when obtained from different dealers and therefore do not necessarily reveal the same structures. Polyclonal antibodies can reveal more; they have a broader detection spectrum. This will result in brighter fluorescence staining but contain an increased possibility of cross-reactions. Phagozytic cells can be stained (nonspecifically) with all antibodies; they can eat everything. The situation with dead cells or debris is similar; they can be stained with everything. Coming to the dyes: Table II lists the most common used dyes in immunofluorescence work together with the laser line used to excite each dye and its emission maximum. The precise spectra can be found elsewhere (Haugland, 1999, or Internet resources). The classic dye fluorescein diacetate (FITC) can be used together with R-Phycoerythrin (PE, a phycobiliprotein, derived from bacteria and algae) and PE-Cy5 (CyChromeTM) for triple staining (with blue excitation) because of their different emission spectra. Disadvantages of this combination are the rapid

TABLE II Some Common Dyes Used in Immunofluorescence Work Dyes, common names/appr. Dyes for multicolor staining Fluorecein isothiocyanate (FITC) Phycoerythrin (PE) PE-Cy5 (CyChromeTM), tandem dye Allophycocyanin (APC) Amino methylcoumarin acetic acid (AMCA) Tetramethylrhodamine isothiocyanate (TRITC) Texas RedTM New developments, dye families Alexa350TM–Alexa594TM BODIPYTM Cy2TM–Cy7TM

Excitation/emission (nm)

488/530 488/575 488/670 633/660 364/455 514/575 568/615

10/31/2000

05:27 PM

262

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

bleaching of FITC together with its strong dependency of fluorescence intensity on pH value and the high molecular weight of PE (∼240 kDa), which makes this dye minimally suitable for intracellular staining. PE-Cy5 is a tandem dye, which is a combination of PE and Cy5 with internal energy transfer. This means, light is absorbed by the PE part of the tandem dye, and energy is transferred radiation-free to the Cy5 part, which emits fluorescent light. The overall result of tandem dyes is a big Strokes shift and the emission of long wavelength light. The disadvantage of this system is the large size of the molecule. Instead of PE-Cy5 the dye PerCP from B/D sometimes is used. This is a chlorophyll protein, which is very photosensitive. It bleaches so rapidly that it can be a problem even in flow cytometry. For using more than three-color staining, the visible optical spectrum will be crowded and a second laser must be used to excite somewere at the end or outside of the visible region. By expanding the optical spectrum to UV and far red, two more dyes became available: allophycocyanin (APC) and amino methylcoumarin (AMCA). APC, which is a member of the phycobiliprotein family, is of interest because of red excitation, making it possible to excite with cheap HeNe lasers or laser diodes, and the low fluorescence background of all biological species illuminated with red light. A disadvantage is the reduced sensitivity of the human eye in the fluorescence maximum; therefore, staining is barely visible in the fluorescence microscope. AMCA, a coumarin derivate, lies on the opposite side of the optical spectrum and must be excited with UV light, which means it requires expensive and power-consuming lasers. Although high background fluorescence is usually associated with UV excitation, this dye works on most samples that contain large numbers of binding sites. Most of the dyes mentioned in this section (with the exception of AMCA, which requires UV excitation) can be excited with Ar- or HeNe lasers, which are easy to handle, stable, and relatively inexpensive. Other available dyes are optimally excited with lasers that are not so easy to handle, such as Krypton or Dye lasers. An example is tetramethylrhodamin-isothiocyanate (TRITC), which was used a lot in early immunofluorescence microscopy. Because of its longer wavelength, TRITC provided a more stable alternative to FITC. It was also commonly used in double staining with FITC. TRIC can be excited (with minor efficiency) with the green line of Ar- or HeNe lasers. The situation is similar with the dye Texas RedTM, the chemical name of which is rather complex, so it’s known by its catchy trademark name. These classic dyes will soon be replaced by some new families of dyes, synthesized and propagated mainly by Molecular Probes. These new dyes should provide greater stability against photobleaching, generally increased fluorescence intensities, and less pH dependency of fluorescence intensities. The first is the AlexaTM family, containing (now) seven members, characterized by a suffix telling the maximum of absorbance. The spectral properties were manipulated by reducing/expanding molecule length, resulting in higher/lower excitation maximum. BodipyTM dyes, which are boron dipyrromethane, compose a family for which

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

263

the spectral properties were similarly manipulated. The advantage is the narrow emission band. The CyTM family comprises cyanine dyes that also cover the whole visible spectral range. All these dyes can be used to stain antibody molecules using some modifications/spacers to prepare reactive groups for convenient conjugation. for a variety of dyes, labeling kits for simple protein labeling are available. New in dye development is the combination of these dyes to tandem dyes; one partner, mostly PE, is used to expand the Strokes shift. Tandem dyes with small molecules for intracellular staining seems not to be available yet. b. DNA, RNA As mentioned earlier, DNA analysis was the first and is still a main application of flow cytometry. Accordingly, a huge amount of literature exists on the subject. Some recent general reviews include Darzynkiewicz and Traganos (1990), Gray et al. (1990), Bagwell et al. (1991), Juan and Darzynkiewicz (1998), and Darzynkiewicz et al. (1999). More clinically orientated reviews include Rabinovitch (1993), Darzynkiewicz (1993), and Lacombe and Belloc (1996). Some fundamental papers on cell cycle analysis and proliferation (Elias, 1997; Ormerod, 1997) and on tumor classification (Tribukait et al., 1986; Petersen and Friedrich, 1986; Bigner et al., 1987) also should be noted here. Functional staining, including live/dead discrimination, apoptosis, and molecular biology applications, together with genetics, will be discussed later in this article. This section concentrates on DNA analysis together with multicolor staining; for example, cell cycle analysis combined with immunological staining. Normally, all eukaryotic cells (and also bacteria and yeast) in G0 and G1 phase of the cell cycle have the same diploid DNA content, sometimes expressed as 2C, or DNA-index equal 1. The DNA-index is defined as the amount of DNA of the respective cell/DNA-content of a normal cell. In an idealized experiment all cells should have the same fluorescence intensity after staining with a DNA staining dye, assuming that the reaction DNA and dye are stoichiometric, meaning that the number of dye molecules is equivalent to the number of DNA molecules. In reality, most experiments show variations due to staining procedure, possible staining of RNA, instrument variation, and variation of DNA content of single cells. Variation is expressed as a coefficient of variance (CV; standard deviation × 100%/mean channel number). The lower the CV of the observed peaks, the greater the amount of information that can be derived from the experiment, and the greater the possible resolution (i.e., narrow peaks will decrease the possibility that two individual peaks will melt and become inseparable). Separation of two populations is very important, because small variations in ploidy (which was defined originally as the number of chromosomes in a cell but now in FC refers to the total amount of DNA per cell regardless of chromosome content and abnormalities) have considerable biological significance. In clinical oncology abnormalities in ploidy were found in malignant tumors, and the pattern of abnormalities can often influence the treatment and the prognosis. Instrument variation as

10/31/2000

05:27 PM

264

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

measured with beads (SectionII.G) should be better than 2%; staining variations should be less than 5% and can be improved by proper dyes and precise staining protocols. Other problems in visualizing separate peaks beside the “normal G0/G1-SG2M-peaks” depend on the possibly overwhelming presence of other fractions and/or on very minor DNA-content differences that merge individual peaks. However, to make a “quick and dirty” decision of cell growth, a simple histogram of stained DNA, separating all cells with fluorescence intensity more than G1, will usually suffice. Guidelines do exist for cell preparation that yield more precise measurements (Fried et al., 1976; Fried et al., 1978; Thornthwaite et al., 1980; Taylor, 1980, Vindelov et al., 1983c; Thornthwaite, 1985; Vindelov and Christensen, 1990). Because of the great importance of DNA measurement in clinical use, some conventions (Hiddemann et al., 1984) were published and a complete issue of Cytometry (Vol. 14, 1993) was dedicated to consensus reviews of this topic: among them, a general article (Hedley et al., 1993), clinical considerations (Shankey et al., 1993a), bladder cancer (Wheeless et al., 1993), breast cancer (Hedley et al., 1993), colorectal cancer (Bauer et al., 1993a), neoplastic hematopathology (Duque et al., 1993), and prostate cancer (Shankey et al., 1993b). Calculation of the number of cells in the G1/S/G2 phase is normally done with more or less expanded curve-fitting programs (ModFitTM, Verity Software House, MulticycleTM, Phoenix Flow System) based on a mathematical model concept (Baisch et al., 1982; Gray and Darzynkiewicz, 1987; Bagwell et al., 1991; Watson, 1992; Bagwell, 1993; Rabinovitch, 1993). These models all have more confidence with lower CV; for proper computing, it helps to know whether there are, for example, tetraploid-contaminating cells, possible aneuploid residues, etc. Pulse shape analysis, like signal processing (as mentioned in Section II.B.4), can be helpful together with multicolor staining of cells to analyze DNA content of interesting cells only. Multicolor staining requires treatment of more or less intact cells. However, if multicolor staining is not required, measurement of nuclei alone is preferable to avoid possible interactions of dye and cellular proteins or RNA. DNA analysis of pure nuclei (membrane and cytoplasm removed by detergents) generally yields precise peaks in a homogenous population. Nuclei from paraffin-embedded tissues (Hedley et al., 1983, 1984, 1985; Stephenson et al., 1986) can be used for retrospective analysis. Those from denatured cells with permeabilized membranes (ethanol may alter some antigens even if they are stained prior to fixation; paraformaldehyde may lead to suboptimal staining profiles) may be used when a lot of dyes are available for multicolor staining. Even live cells prepared with membrane-crossing dyes may be used. These have the added advantage of permitting correlation of functional staining with cell cycle analysis. Turning now to DNA-staining probes, the following discussion will be limited to nonsequence-specific probes. Sequence-specific dyes will be discussed in Section III.B.5.c. Propidium Iodide, (PI, introduced by Crissman and Steinkamp, 1973), with properties similar to the well-known ethidium bromide (EB), intercalates between

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

265

bases in double stranded nucleic acids. Some older, methodological articles include Crissman and Tobey (1974), Krishan (1975), Fried et al. (1978), Taylor (1980), and Vindelov et al. (1983a). PI can be excited with UV and blue light. Its emission spectrum is very broad yellow-red, which makes it the dye of choice for single color staining, but it requires special attention for multicolor staining due to overlapping of its spectrum with the spectra of most other dyes. To obtain good resolution, double-stranded RNA must be removed with RNAse or by detergent-lyses of the cell (Vindelov et al., 1983b). PI cannot pass through the intact cell membrane, so cell membranes must be permeabilized before staining. As will be discussed later on, PI is a very useful discriminator between live and dead cells. In addition, staining equilibrium occurs within minutes. DAPI 4 -6 -diamidino-2-phenylindole, was first applied to mycoplasma detection by Russell et al. (1975). It is A-T specific, excited by UV light, and emits blue light (Thornthwaite et al., 1980; Kapuscinski, 1995). DAPI seems to have the lowest CV of all DNA-specific dyes and is therefore the dye of choice for instruments equipped with an UV laser (Otto, 1994). DAPI staining is assumed to be less affected by the state of chromatin condensation than is staining with other DNA stains (Darzynkiewicz et al., 1984; Evenson et al., 1986). As with PI, DAPI cannot cross intact cell membranes. 7-ADD, 7-aminoactinomycin D, is G-C specific, and its excitation/emission spectra are on the other end of the optical spectrum (Gill et al., 1975; Zelenin et al., 1984). 7-AAD can be excited with blue-green light and emits red. Despite its drawbacks—low quantum efficiency and fluorescence intensity correspondingly dim, CV broad because dye-binding is affected by chromatin structure (Stokke and Stehen, 1987)—7-AAD is an useful dye for multicolor staining and for live/dead cell discrimination because it does not interfere in its spectrum with other dyes for immunological staining (Rabinovitch et al., 1986). Hoechst 33342, one of several bisbenzimidazole dyes with different numbers (e.g., Hoechst 33258, which has a slightly slower cell permeability as shown by Arndt-Jovin and Jovin, 1989), is the classic dye used for staining live cells (Latt and Stetten, 1976; Arndt-Jovin and Jovin, 1977; Hamori et al., 1980; Loken 1980a). It must be excited with UV, emits blue and minor red light, and binds to A-T rich regions in the small groove of DNA. The uptake rate of viable cells is dependent on experimental conditions and cell type, so differences between cell type and cell status (see also Section III.B.4.a) can be demonstrated (Lalande and Miller, 1979; Loken 1980b; Williams et al., 1982). As Hoechst 33342 appears to be nontoxic, it also can be used for cell tracking (see Section II.B.4.b). Mithramycin, Chromomycin A, and a few other similar antibiotics are G-C specific with excitation maxima mostly in the violet portion of the spectrum. They emit green light (Crissman et al., 1978; Crissman and Tobey, 1990). Newly developed cyanine dyes TOTO (two molecules of thiazole orange linked with dizaundecamethylene) and YOYO (two molecules of oxazole yellow, similary liked) are both cell-impermeable (Hirons et al., 1994; Marie et al., 1996). These dyes are mainly used in gel biochemistry because of their highly sensitive detection

10/31/2000

05:27 PM

266

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

of nucleic acids (Benson et al., 1993a, 1993b). They are not as useful in flow cytometry because of their slow penetration, even in permeabilized cells. SYTO is a whole family of dyes, some of which can cross intact cell membranes and some of which stain structures other than DNA, such as cytoplasm or mitochondria. Additional new dyes come from the development of new dye lasers; some can stain nucleic acids. This may prove of interest during the next few years, especially for red excitation in multicolor staining, as is well on the way to be developed for immunofluorescence dyes. A number of authors have conducted comparative studies on DNA stains, describing energy transfer between stains, and alterations in chromatin organization, which tends to affect DNA staining (Otto and Tsou, 1985; Darzynkiewicz et al., 1984; Everson et al., 1986; Stokke and Steen, 1987; Crissman and Steinkamp, 1993; Ashbury et al., 1996; Guindulain et al., 1997; Mason et al., 1998; Lebaron et al., 1998; Abdullah et al., 1999). In general, dyes with different base preferences have been found to give varied results in staining of chromosomes with different base composition, an effect discussed later in Section III.B.5.b. All the dyes mentioned here seem to stain not only DNA but also to stain double-stranded RNA to varying degrees. PI, for example, is reported to be a useful RNA stain when combined with DNAse to avoid DNA staining. A dye that stains mainly double-stranded (ribosomal) RNA is Thioflavin T, a basic diazole dye (Arndt-Jovin, 1990) that is better known for staining reticulocytes (Tanke et al., 1981). Another known dye is Pyronine Y, a xanthene homologue of Acridine Orange. It can enter living cells, stain double-stranded DNA and RNA, and sometimes stain mitochondria. However, it is toxic in working concentrations needed for staining (Shapiro, 1981; Crissman et al., 1985; Darzynkiewicz et al., 1987). Double-staining studies involving saturation of DNA with Hoechst33342 prior to pyronine Y staining have permitted calculations of DNA and RNA of live cells. Acridine Orange, (AO) is an almost mystic dye in the field of flow cytometry. AO is difficult to handle because of its sensitivity to very slight changes in staining procedures (Darzynkiewicz et al., 1980). Under proper acid conditions and with a very critical concentration, it intercalates into double-stranded intact DNA, forming monomeric green fluorescent complexes (after blue excitation). All RNA present is converted to the single-stranded form and stained with an AO dye polymer that emits red fluorescence light. Using this method, DNA and RNA content can be measured simultaneously (Traganos et al., 1977; Grunwald, 1993; Darzynkiewicz, 1994). Some additional remarks follow on cell cycle analysis. A series of cellular processes that preceed cell cycle can be analyzed by flow cytometry. The best known is the bromodeoxyuridine (BrdU) method of measuring DNA synthesis. BrdU is an analog of thymidine and competes with that base for during synthesis of DNA. After incorporation, it can be detected by a monoclonal antibody (Gratzner et al., 1975; Gratzner, 1982; Dolbeare et al., 1983). In double-staining studies with a DNA stain, the percentage of G0/G1-S-G2/M can be determined without

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

267

mathematical curve-fitting programs. Some newer reviews on this methodology include Poot et al. (1994), Dolbeare (1995). A different approach to cell cycle studies is the detection of nuclear antigens, among them cyclins (Gong et al., 1993, 1994) and proliferating cell nuclear antigen (PCNA, Celis et al., 1984; Kurki et al. 1986, Teague and El-Naggar 1994). c. Protein The total protein content of cells is not considered as important nowadays as it was years ago. Today, more or less specific probes are available (for example monoclonal antibodies) for detection of epitopes as small as a few amino acid sequences. The following reviews provide an overview of total protein content as a measure of growth and metabolism of mixed cell populations: mainly in double staining with a DNA-stain (Crissman et al., 1981, 1985); nuclear protein together with cell cycle (Roti Roti et al., 1982; Auer et al., 1983). d. Carbohydrate, Sugar, Lipids, Cholesterol The Schiff-reaction exists for detection of total cellular carbohydrate content in a cell (Duijndam and van Duijn, 1988). Various other techniques permit measurement of more specific lectinbinding sites (Kraemer et al., 1973; Steinkamp and Kraemer, 1974). Interest in lectin-binding sites has been on the decline since the advent of monoclonal antibodies (Bauman et al., 1988). Nevertheless, a series of interesting applications have been described that mainly differentiate between subpopulations of cells on the basis of lectin binding. Examples include nucleated and non-nucleated erythrocytes, cell differentiation, neoplastic transformation, and subcellular organelles (Guasch et al., 1993, 1995). Membrane-staining dyes first spring to mind when discussing lipids. Various probes exist for staining cell membrane at various depths. The best known is diphenylhexatriene (DPH), which is used for membrane microviscosity measurements. (This will be discussed in more detail in Section III.B.4.g.) Filipin forms a fluorescent complex with cholesterol (Muller et al., 1984: Hassal and Graham, 1995) and can therefore detect foam cells (Hassall, 1992). Lipoprotein binding and metabolism (LDL and HDL) was investigated by various authors (Traill et al., 1987a; Traill et al., 1987b; Schmitz et al., 1987; Huber et al., 1990; De Sanctis et al., 1995; Maczek et al., 1996; Streicher et al., 1999). e. Energy Transfer, Proximity Fluorescence resonance energy transfer (FRET) is used in flow cytometry in tandem dyes as mentioned in Section III.B.3.a. The physical principle is the non-radiative transfer of energy from a donor molecule (molecule excited by light) to a second molecule (acceptor), the light emission of which can be observed without direct excitation (Szollosi et al., 1984, 1987, 1998). The efficiency of this process depends on several dye properties, mainly the distance between the two molecules (in the nanomolar range). Energy transfer has found broad application as a “molecular ruler” for measuring cell receptor proximity (Stryer and Haugland, 1967). First introduced in flow

10/31/2000

05:27 PM

Cytology

PS012-05.tex

268

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

cytometry to demonstrate the proximity of lectin receptors (Chan et al., 1979), it is now used for investigating receptor associations and for determining whether two different ligands (mainly monoclonal antibodies) bind to the same receptor (Tron et al., 1987; Szollosi et al., 1989; Harel-Bellan et al., 1990; Matyus, 1992; Matko and Edidin, 1997). In molecular biology, FRET is used in connection with PCR. 4. Functional Staining Most cellular functions are time dependent. Some cells have a very quick response to external stimuli, and some are stongly dependent on experimental conditions such as temperature. A few technical systems exist for catching these rapid responses. One such system involves mixing stimulating substances with cells immediately before measurement, together with heating devices (Kachel et al., 1982; Omann et al., 1985; Kelley, 1989; Kelley, 1991). For slower activities such as phagocytosis, almost all commercial cytometers can use time-dependent acquisition of data. a. Live/Dead, Apoptosis Cell viability, membrane integrity, and cytotoxicity are more complex than is generally believed. When referring to cell cultures, the term viable usually means that cells are functional (reproductive, etc.). But in flow cytometry, viable is mainly defined as “membrane integrity,” and various dyes exist that cannot cross intact cell membrane and thus can stain only “dead” cells. This is analogous to the well-known trypan blue exclusion test used in microscopy. However, controversial studies with lethal doses of drugs (Bhuyan et al., 1976; Roper and Drewinko, 1976) have shown that some cells that can exclude dyes are not viable. Controversely, cells can have a loss of membrane integrity without being dead. This forms the basis of electroporation, which involves punching holes into cell membrane; cells should survive this procedure at least for some time. All cell membrane-impermeant DNA staining dyes mentioned in Section III.B.3.b. are more or less suitable to serve as a dye excluding live/dead detector. Even the Hoechst dyes, which are able to cross intact cell membranes, can be used as such because the dye will stain nuclei alone within seconds, whereas it will take about an hour to reach equilibrium in intact cells. As a rule of thumb, for simple live/dead information in multicolor staining experiments, the dye with the smallest overlap with other stains is the dye of choice (e.g., Schmid et al., 1992; O’Brien and Bolton, 1995; Barber et al., 1999). For fixed samples, staining with ethidium monoazide (EMA) is done before fixation. EMA can be covalently crosslinked to nuclear acids by visible and UV light, thus providing a stable marker for cells that were damaged before fixation (Riedy et al., 1991). In addition to the large number of dyes available for use in dye-excluding tests for viability (among them new dyes from Molecular Probes, SYTOX, where X is a number or character), a large number of dyes have been developed for use in dye-including tests and protocols. Most stain various cell functions. A typical,

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

269

classical example is fluorescindiacetate (FDA), which is nonfluorescent, can cross intact cell membranes, and is hydrolyzed inside the cell by a nonspecific esterase that is present in almost all cells (Rotmann and Papermaster, 1966). The effect is two-fold: the resulting molecule is highly fluorescent, and it is unable to cross the cell membrane. Many new dyes trace cell function other than enzyme activity, including membrane potential, changes in intracellular radicals, etc. An overview is given in the Molecular Probes handbook (Haugland, 1999). All of them will stain viable cells because of some functions, so the investigator can define which cells are alive and working and which are dead or quiet for her or his own experiment. Similar to this live/dead discrimination is the situation in apoptosis detection. Apoptosis, or programmed cell death, is of great interest in many disciplines, particulary cancer and developmental research. A great deal of literature therefore exists on the topic (recent reviews include Darzynkiewicz et al., 1994; Lacombe and Belloc, 1996; Loo and Rillema, 1998; Moore et al., 1998; Ormerod, 1998; Burchiel et al., 1999). Apoptosis was originally defined as fragmentation of DNA due to activation of endonuclease is a multistep process. Not only enzyme activities, but also alterations in cell membrane composition, mitochondria, many cellular functions, and morphological changes are detectable in FC. Morphological changes of apoptotic nuclei often can be seen by alterations in scatter signals (Darzynkiewicz et al., 1992), and fragmentation of DNA can be measured by all DNA-staining dyes, the most popular of which is PI (Nicoletti et al., 1991). DNA loss results in a nucleus with reduced fluorescence intensity compared to G0/G1 (“sub-G0 peak”). Fragmentation can be observed directly by the TUNEL (terminal deoxynucleotidyl transferase, TdT) method, which labels strand breaks (Sgonc and Gruber, 1998). Membrane changes can be detected by the translocation of phosphatidylserine (PS) from the inner layer of the cell membrane to the outer layer (van Engeland et al., 1998). Additional cell membrane changes can make the nuclei more accessible to DNA-dyes (e.g. Hoechst33342), so DNA of an apoptotic cell can be stained with an appropriate dye concentration. There are virtually a couple hundred methods available to detect functional changes in apoptotic cells, most of them correlated with mitochondrial alterations (Poot et al. 1997), surface antigens changes, or cell cycle analysis in multicolor staining (Sgonc et al., 1994; Darzynkiewicz et al., 1998). All the processes, followed by fluorescence labeling, are time dependent; some are of short duration and more or less pronounced. Changes seen with one detection method within a certain time scale are not necessarily seen with other methods. It is therefore important to know what the respective cells do in a particular experimental situation and to know the corresponding time scale of cell actions in order to determine the best mechanism for detection of apoptosis in that system. b. Phagocytosis, Cell Tracking The most direct way to detect phagocytosis is to offer the phagocyte stained particles, usually macromolecules, such as dye-labeled dextran, polystyrene particles in the form of labeled microspheres, or stained

10/31/2000

05:27 PM

270

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

bacteria or yeast (Steinkamp, 1982; Oda and Maeda, 1986). All these particles are available in a wide range of size, shape, and antigenicities, as well as opsonized, and are labeled with various dyes as used in immunofluorescent staining. A second dye, known as an acceptor, may be applied to induce FRET and help determine whether the particle is really internalized or only attached to the outside of the phagocyte cell membrane. Alternatively, and more simply, an unspecific fluorescence quencher such as trypan blue (Sahlin et al., 1983; Bjerknes and Bassoe, 1984) may be included to prevent (“quench”) fluorescence from nonspecifically attached particles. One important application of cell tracking is detection and following of stained bacteria after phagocytosis. Some other applications are detection of cell hybridization (where two cell types with different membrane staining can be identified after fusion by their mixed color fluorescence or by energy transfer between tracking dyes), localization of cells after injection in animals, or estimation of cell proliferation rate (Lyons, 1999). DNA staining also can be used to estimate the rate of cell proliferation; because daughter cells share DNA content, they will be less bright. However, the best way is to stain cell membrane (not the DNA), since cell function is only slightly affected even when staining is heavy. Dyes suitable for this purpose are all lipid stains. They include dioctadecylindocarbocyanine (DiI) and similar substances such as DiO or DiU and dyes of the PKH family (Horan et al., 1990; Ford et al., 1996; and Parish, 1999). PKH seems to penetrate deeper in cell membrane and will therefore be more persistent than the others. c. pH Variations in pH, observed mainly in neutral or more acid organelles (first by Visser et al., 1979), seem to correlate with various cell functions and alterations, among them cell stimulation (Gerson et al., 1982) and malignancy (Valet et al., 1981). As mentioned earlier, the fluorescence intensity of FITC is highly dependent on pH, thus making FITC a good dye for follow variations in intracellular pH (Musgrove, 1986). Another commonly used dye is SNARFTM, with constant blue excitation, the dye shifts its emission spectra from orange to red under increasing basic conditions (Haugland, 1999). BCECF (2 7 -bis-carboxyethyl5,6-carboxyfluorescein) shows pH-related variations in its excitation spectrum. Under basic conditions, its absorption rate increases, shifting from violet to blue (Haugland, 1999). d. Ions There is considerable interest in ions other than those known to regulate pH. Ca2+ -flux measurements are used routinely to study signal transduction in different cell types after stimulation with specific agents. Depending on the technique chosen, Ca2+ -flux may be expressed as a ratio of the excitation or emission wavelength shift (Grynkiewicz et al., 1985; Valet et al., 1985) or as an increase in the fluorescence intensity (fluo-3, described first by Minta et al., 1989). The respective dyes are: excitation wavelength shift, Fura-2 (less suitable for flow cytometry because its excitation spectrum does not match a convenient laser line);

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

271

emission wavelength shift, Indo-1. Ratiometric measurements have the advantage of cancelling out the effect of variations of individual cells in dye content, but the disadvantage is that such staining requires almost the whole spectrum, so multicolor staining needs a multilaser instrument. A rarely mentioned side effect seems to be the long-term toxicity of Ca2+ staining dyes, mainly after observation (i.e., after light absorbing; G. Boeck, unpublished observation). Overviews of calcium ion labels have been published by Kao (1994), June and Rabinovitch, (1994), and Silver (1998), and on multicolor staining by Rabinovitch et al. (1986) and Lazzari et al. (1986). Excellent overviews of methods and appropriate dyes were written by Scheenen et al. (1998) and Haugland (1999). Another metal ion of particular interest is Mg2+ , mainly because of its regulatory role in enzymatic reactions and hormone secretions. As for Ca2+ indicators, several dyes exist, some of which increase fluorescence intensities after binding (Magnesium Green®), and change excitation/emission spectra (Magindo-1). Dyes detecting Mg2+ are structured very similarly to those for detecting Ca2+ , and in fact there is some overlap: Ca2+ can disturb Mg2+ measurements. Similarities also exist between detection of other metal ions, among them Zn2+ , Cu2+ , Cd2+ , Hg2+ , Ni2+ , Al3+ and others (Haugland, 1999). Most of them can be detected by Ca-staining dyes. Physiological changes in Na+ and K+ level can be measured ratiometrically by flow cytometry with the benzofuranyl fluorophores SBFI (Minta and Tsien, 1989), and PBFI (Meuwis et al., 1995), respectively, or with Sodium Green™(Haugland, 1999). The intracellular content of Cl− ions can be measured with SPQ [6-methoxyN-(3-sulfopropyl)quinolinium] (Wolfbeis and Urbano, 1982; Illsley and Verkman, 1987) with some (quenching) interference to other halide ions such as Br− and I− . More information on these stains and on nitrite, sulfite, and phosphate detection can be found in the handbook of Fluorescent Probes (Haugland, 1999). e. Membrane Potential Changes in membrane potential are mainly due to Na+ and/or K+ shifts across the cell membrane. Most of these changes involve Ca2+ flux and/or pH changes, so these processes can be followed as described above. Potential differences across the membranes of mitochondria will be summarized in Section III.B.4.h., so the remaining dyes to mention here are carbocyanine dyes such as DiOCn (Hoffman and Laris, 1974; Sims et al., 1974; Shapiro, 1994), which are dyes decreasing fluorescence intensity upon membrane hyperpolarization, or JC-1 (Reers et al., 1991; Smiley et al., 1991), which is usable for ratio measurements. Oxonol compounds are more specific in terms of reduced staining of mitochondria, but the specific staining is weak and disturbed by bright dead cells (Wilson et al., 1985; Wilson and Chused, 1985). Since all of these membrane-potential and iondetecting dyes cause either some disturbance of normal cell metabolism or bind nonspecifically to DNA, long time studies must be approched with care (Latt et al., 1984; G. Boeck, unpublished personal observations).

10/31/2000

05:27 PM

272

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

f. Oxidatives, Enzymes Enzyme and/or oxidative reactions originally were used in FC to detect functional changes and dead cells, respectively. It is still common today to follow oxidative metabolism of phagozytic cells: the nonfluorescent DCFH-DA (2,7-dichlorofluorescin diacetate) can penetrate the cell membrane and is cleaved by nonspecific esterases to DCFH, still nonfluorescent, but membrane impermeable. In the presence of peroxide and H2 O2 , formed during a respiratory burst, DCFH is converted to the fluorescent DCF (2,7-dichlorofluorescein), as shown by Bass et al. (1983) and Severin and Stellmach (1984). This method can be used to quantify respiratory bursts following phagozytosis in blood neutrophils or monocytes (Trinkle et al., 1987; Burow and Valet, 1987; Perticarari et al., 1991). Monocytes give a weaker reaction than neutrophils because of their lower peroxides content (Casado et al., 1993). The immune status of individual cells can be estimated (Helmberg et al., 1989). A similar method with the same dye seems to work for detecting nitric oxide (Rao et al., 1992). Dihydroethidium (hydroethidine®, HE) enters live cells and is oxidized to ethidium, staining double-stranded nucleic acids (see also Section III.B.3.b). The fluorescence is affected by superoxide. So both of these oxidative reactions can be demonstrated simultaneously in a double-staining experiment (Rothe and Valet 1990). Glutathione levels, which are of interest because of their involvement in drug resistance (Rice et al., 1986) and infection development (Roederer et al., 1991), can be examined by various dyes, among them bromobimane (Hedley and Chow 1994), o-phthaldialdehyde (OPT, Treumer and Valet 1986), and mercury orange (O’Connor et al., 1988). Oxidative enzymes are detectable without fluorescence staining: nitroblue tetrazolium (NBT) can form an amorphe precipitate, thereby disturbing normal forward and side scatter signals (Blair et al., 1985). Peptidases and acid phosphatase activities were detected by various dyes (Watson 1980; Dive et al., 1987). A word of caution should be mentioned here. Because enzyme activities are strongly timedependent, as are other functions such as changes in pH and ions, the multiparameter measurements of interest may follow a completely different time scale or, even worse, may disturb each other. g. Fluorescence Polarization, Microviscosity As most of the lasers used in FC emit polarized light, fluorescence polarization measurements can easily be performed by adding polarizing filters in the emission light path. The principle is that dye molecules have a distribution of charges; in other words, they are polarized. This means that there is a probability of polarized light being absorbed, depending on the direction of excitation. Because probe molecules can move (for example, rotate) and there is a time interval between light absorption and fluorescence emission, emitted fluorescence will be in different directions from light polarization. If the probe molecule is in a viscous environment or is fixed on a surface, it will rotate slowly and the polarization of the emitted fluorescence will be more similar to the direction of excitation polarization. Depolarization of fluorescence emission

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

273

therefore provides a measure of the mobility of the probe molecule (Shinitzky and Inbar, 1976). Measurements of the polarization of fluorescence emission have often been used to monitor the physical environment of the probe molecule, mainly in cell membranes. Various dyes can be used as probe molecules in different membrane layers: 1,3,5-diphenyl 1,6-hexatriene (DPH) has been reported to stick in the outer lipid layer of the membrane, penetrating only slowly into the inner layer. Trimethylammonium-DPH (TMA-DPH) may be more stable than DPH, with the dipole stacking in parallel to the long molecular axes of the lipid molecules. This method of fluorescence depolarization has been used to demonstrate differences in the membranes of human white blood cells from young and elderly people (Boeck et al., 1989; Collins et al., 1991) and between normal and leukemia cells (Ben-Bassat et al., 1977). These differences correlate with the cholesterol content of the cell membranes (Shinitzky and Henkart, 1979) or with multidrug resistance (Dudeja et al., 1995). h. Organelles Inside the cell membrane, a variety of interesting structures can be analyzed in flow cytometry, among them DNA/RNA (see Section III.B.3.b) and proteins that can be detected by antibodies after membrane permeabilization. Beside the measurement of these structures, the fluorescence analysis of intracellular organelles is of increasing interest mainly in respect to functional and/or biochemical analysis in multicolor staining. FC not only provides a powerful tool for the analysis of living cells, but also for sorting pure samples of organelles for biochemical/molecular biological analysis or for in vitro studies. An overview of some of the more important intracellular applications of FC is given in Table III. Purification of Mallory bodies, stained with a monoclonal antibody and sorted for further biochemical analysis, should stand here as an example for analysis and enrichment of antibody reachable proteins/particles (Zatloukal et al., 1991). A similar approach works for the proteins of the Golgi complex. For example, an antibody raised against glycosyltransferases can be used for analysis or with fluorescein-conjugated concanavalin A (FITC-ConA) to study cis and trans fractions (Guasch et al., 1993, 1995). The Golgi apparatus can be selectively stained with 6-[(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl] sphingosine (NBD C6-ceramide) and BODIPY FL C5-ceramide (Pagano et al., 1991, Pagano and Martin, 1998) together to monitor secretory activities (Ktistakis et al., 1995). The Golgi complex and the endoplasmic reticulum (ER) can be stained with BODIPY-brefeldin A (Deng et al., 1995). The ER and some nuclear membrane can be stained with certain textile dyes (Simmons et al., 1990). Endocytic compartments can be accessed from the outside of the cell either with labeled ligands of internalizing receptors or with membrane-staining fluorescent dyes that are taken up by endocytosis (Wilson and Murphy, 1989). Studies of binding, uptake, acidification and degradation of ligands were performed with

Labeling in brain homogenate

Vital stains Vital stains

Nondenaturing purification of tangles

Depend on mitochondrial membrane potential Independent of membrane potential

Degradation of fluorescent substrate by cathepsin B Preparative sorting of endosomes

Preparative sorting of endosomes,

LaTeX2e(2000/08/16)

Tableadapted from Guenther Boeck, Peter Steinlein, and Lukas Huber (1997), Cell biologists sort things out: Analysis and purification of intracellular organelles by flow cytometry. Trends in Cell Biology 7, 501, with permission from Elsevier Science.

Congo red

Neurofibrillary tangles

Fluid-phase endocytosis

N-Carbobenzyloxy-ala-arg-4methoxy -naphthylamine TMA-DPH Rhodamine 123, JC-1 Nonyl acridine orange

Fluid-phase endocytosis Characterization of water channels

6-Carboxyfluorescein

Preparative sorting of endosomes, receptor

PS012-05.xml

Mitochondria

Receptor-mediated endocytosis regulation

FITC-prolactin

Stain nuclear membrane and ER Identification of endocytic mutants Binding, uptake, acidification, degradation

Purification of MB Protein analysis Separation of cis and trans membranes Visualize Golgi and ER

Analysis

PS012-05.tex

Intercalates into plasma membrane endocytosis

Vital stains Fluid-phase endocytosis

Textile dyes FITC-dextran

Endoplasmic reticulum (ER) Endosomes/lysosomes

Labeling in liver homogenate Labeling of lymphoid cells Binding to isolated stacks Labeling of living cells

Labeling

Ab against MB Ab against glycosyltransferases FITC-lectins BODIPY-brefeldin A

Fluorescent probe

Cytology

Mallory bodies (MB) Golgi

Organelle/cellular component

05:27 PM

TABLE III Intracellular Applications of Flow Cytometry

10/31/2000 Textures 2.0

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

275

advanced fluorimetric methods (Lane and Chen, 1991; Chang et al., 1994). Flow cytometric sorting and enrichment of stained particles for further cell biological analysis was done with various staining techniques and organelles (Murphy, 1985; van der Goot et al., 1992; Boeck et al., 1998). Mitochondria appear to be the main topic of most publications on organelle work (Poot, 1998). The classic dye Rhodamine 123 (Chen, 1989) and the newer ratiometric probe JC-1 (Cossarizza et al., 1993) can monitor mitochondrial membrane potential. They have been applied in various fields of interest, among them observation of isolated individual mitochondria (Cossarizza et al., 1996), sensitive detection of apoptosis and necrosis (Petit et al., 1995; Bedner et al., 1999), and following drugs resistance (Mancini et al., 1998). Carbocyanine dyes such as DiOC6 have been reported to stain mitochondria in algae (Suzuki et al., 1994) and plant cells (Liu et al., 1987). The uptake of another dye, nonyl acridine orange, is independent on mitochondrial membrane potential (Maftah et al., 1989). It is used to follow apoptosis (Ferlini et al., 1996). More information about organelle-specific dyes can be found in the review of Plasek and Sigler (1996), and in the handbook of Molecular Probes (Haugland, 1999). Among them is a whole new family of probes called MitoTracker. These dyes are available in various fluorescent colors together with various derivates. Staining with Congo Red and further enrichment of neurofibrillary tangles surves as a good example of the use of flow cytometry in biochemistry purification (Hussey et al., 1986). Many of the above mentioned dyes have been investigated in microscopy or microfluorimetric work, together with “unusual” (in terms of function) cell systems. Specificity seems to be the main drawback of many of these dyes. It is essential to establish specifity for each different probe before proceeding to regular flow cytometric work. 5. From Cell to Molecule FC multicolor analysis and sorting is finding increasing application in the field of molecular biology. Flow cytometer instruments are becoming bigger as they are used for analysis of smaller structures, even for organelles. Powerful lasers with greater stability, optimized instrumental set-up, stable liquid flow, and low analysis speed help ensure low coefficient of variation. a. GFP The green fluorescence protein, GFP, from the jellyfish Aequorea victoria has in a short space of time become an important probe for marking and visualizing cells and proteins (introduced by Calfie et al., 1994) and for following gene expression in a variety of cell types (recent review: Tsien, 1998). After incorporation of a GFP expression plasmid into a cell by transfection, the protein is formed by the cell metabolism without any additional cofactors, so the staining lies somewhere between intrinsic and extrinsic. Despite the drawback of weak

10/31/2000

05:27 PM

276

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

fluorescence encoded from the wild-type GFP gene, the advantages are impressive. GFP is a very stable molecule, it fluoresces green after absorbing violet or UV light, and its expression is species independent (Ropp et al., 1995). Its possible toxicity seems to be a point of controversy (Liu et al., 1999). Extensive studies of the protein, together with the sequencing of the cDNA, have led to a variety of applications. Mutants have been found to enhance the intensity of staining (Helm et al., 1995; Cormack et al., 1996). Proteins with spectral properties different from the original (BFP, blue fluorescence protein; YFP, yellow fluorescence protein; Zhu et al., 1999; Beavis and Kalejta, 1999) allow multicolor staining. The rapid expansion of literature in this area mirrors the increasing interest in this method; for example, in leukemia work (Chu et al., 1999), for detection of expression from two different genes within single mammalian cells (Anderson et al., 1996), and for identification of transfected cells together with functional staining (Demo et al., 1999). Intracellular staining seems to be dependent on use of the proper permeabilization and fixation methods (Kalejta et al., 1997). FC-detection of single copies of GFP so far has yielded strange results (Moerner et al., 1999). b. Chromosomes and PCR Analysis and enrichment of chromosomes by FC and subsequent construction of DNA libraries turned out to be a powerful tool in molecular biology. Starting with single staining with a DNA specific dye such as PI, the contents of DNA per chromosome were analyzed in monovariate histograms (as demonstrated in Fig. 1b). Studying DNA staining dyes, it was found that some dyes are sensitive to DNA base composition: Hoechst 33258 and DAPI (see Section III.B.3.b) are A-T specific; Chromomycin and Mitramycin are G-C specific. In the human genome (but not in all organisms, e.g., bacteria), the overall A-T content is equal to the G-C content, but single chromosomes can be classified by their differences in A-T and G-C content (van den Engh et al., 1986). This leads to double staining of chromosomes were genetic abnormalities can be found by shifting a peak to higher or lower fluorescence intensities; a lower limit of detection of about two mega-base-pairs have been reported (Trask et al., 1996). Translocations with the same G-C and A-T content will be unrecognized, and the DNA index is needed to detect ploidy. Although chromosomes are stable, the preparation/isolation is not trivial (some protocols: Gray, 1989); this places considerable requirements on the flow cytometer (Van Dilla et al., 1983). Nevertheless, sorting of chromosomes, that is, the isolation of pure chromosome fractions for construction of chromosome-specific gene libraries (for example, Y chromosome, by Fantes et al., 1983), started with high-speed sorters (Peters et al., 1985; Gray et al., 1987). Several hundred thousand chromosomes are needed for establishing libraries, so some preselection of chromosomes (e.g., density centrifugation or selection of appropriate cells with increased proportion of chromosomes in question) is required even with high-speed sorters. The chromosomes of a large number of species have been

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

277

analyzed (Ferguson-Smith, 1997). Semiautomatic methods for detection of aberrant chromosomes have been developed, with sorting and analyzing of questionable chromosomes (Nizetic et al., 1991). DNA amplification by polymerase chain reaction (PCR) was a breakthrough in cytogenetics because the amount of DNA and RNA can be enhanced. To establish libraries today, the lower limit seems to be less than one thousand sorted chromosomes and subsequent PCR. Gaining sufficient for a library is now much more feasible than before (some reviews about chromosome sorting and DNA library production: Cram, 1990; Green, 1990; Metezeau et al., 1993; Fantes et al., 1994). Although PCR on single cells is tricky and results produced may contain artifacts, this remains a powerful method for analysis of rare cells. Sorting rare cells by FC and subsequent PCR (single cell/multitube PCR) is applied in analysis of aberrant chromosomes (Gray et al., 1991). Other successful applications have been reported in follow-up of chromosomes in farm animals (Dixon et al., 1992), localization of breakpoints in human chromosomes (Look, 1988) together with human genetic mapping (Green, 1990; Cram, 1990), the human genome project (Roslaniec et al., 1997), and in clinical diagnosis (Bartholdi, 1990). Despite ethical issues, FC seems to be the (only) technique for preconceptional sex selection by staining of chromosomes with DNA-dyes (Johnson 1995, 1997; Hossain et al., 1998) and subsequent sorting. c. FISH and Chromosome Painting Fluorescence in situ hybridization (FISH) (Trask et al., 1991) has become an analytical tool used more in fluorescence microscopy/image analysis than in FC. Considerable effort has been expended to optimize DNA and RNA preparation and expand the boarders of sensitivity and specificity (some recent reviews: Ferguson-Smith, 1997; in clinical work— Blennow et al., 1992; Brandt et al., 1993; Qian et al., 1996). Without going into details, two types of FISH probes have been established: repetitive sequence probes and sequence specific probes, which are used for painting. The lower limit seems to be only a few amino acid sequences. Staining is done by biotin labeling or by modified DNA (e.g., digoxigenin) together with stained monoclonal antibodies. Bottiroli et al. (1992) demonstrated the use of FRET for in situ hybridization. The main effort of FC in gene characterization is the sorting of cells (after immunofluorescence staining) combined with PCR to characterize sorted cells and/or produce sequence specific probes (Weier et al., 1994; Morgan and Pratt, 1998) with some barriers in doing PCR in suspension (Timm et al., 1995) FC is not routinely used for FISH, because of weak staining with wide variations due to cell cycle stage and permeability of the probe. Sometimes ratiometric staining with others than the requested probes may help, but only a few laboratories seem to be able to do such analysis, sometimes together with pattern recognition systems (slit scan cameras, as mentioned in Section II.B.4).

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

278

Textures 2.0

GUENTHER BOECK

IV. Internet Resources: Flowing along the Net The Internet is gaining increasing importance in biomedical sciences (Dow, 1998). Progress in computer technology and data handling, together with the readiness of researchers to share their expertise with all interested users, has led to an incredible amount of information on the Net. I do not want to go into details here on Internet search strategies, Internet connection, browsers, etc. These can be found at your local computer center or on the Net itself; for example, “The Big Dummy’s Guide to the Internet” (http://www.td.anl.gov/InternetGuide.html). The sites listed in Table IV may provide a good starting point. However, it is is in the nature of the Internet that some of these sites will already have moved or shut down. Fortunately, all of the sites are linked, so finding one will lead the interested reader to the others. Using general search engines has advantages (e.g., new offers can be found) and disadvantages (a lot of not working Universal Resource Locators (URL) together with many unwanted hits). Searching for more specific information with more

TABLE IV Internet Resources for Flow Cytometry: List of URLs General search engines

URL

Alta Vista Lycos Yahoo Magellan Webcrawler

http://www.altavista.com http://www.lycos.com/ http://yahoo.com/ http://mckinley.com/ http://www.crawler.de/

Experts Salk Inst. La Jolla Boston Users Group Univ. Massachusetts Amherst Univ. Texas Med. Branch Max Planck Inst. Germany Mary Hitchcock Mem. Hosp. Geoff Osborne John Curtin Purdue Univ., Lafayette Imperial Cancer Res. Fund Univ. Washington

URL http://pingu.salk.edu/fcm/sitelink.html http://www1.shore.net/∼bugbytes/ http://www.bio.umass.edu/mcbfacs/flowcat.html http://stem.utmb.edu/ http://www.biochem.mpg.de/research-groups/valet/cytorel.html http://130.189.200.66/ProcMan/ http://jcsmr.anu.edu.au/facshome.html http://facs.scripps.edu/protocols.html http://www.icnet.uk/axp/facs/davies/links.html http://nucleus.immunol.washington.edu/Research facilities/ cell analysis.html

Companies Wiley cytometry website Molecular Probes Becton/Dickinson homepage Beckman/Coulter Oriel Amersham

URL http://www.wiley.com/products/subject/life/cytometry/ http://www.probes.com/ http://www.bdfacs.com/ http://www.beckmancoulter.com/ http://www.oriel.com/ http://www.amersham.com/

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

279

specific keywords will result in URL’s of universities, flow cytometry centers, hospitals, societies for flow cytometry, expert centers, newsgroups, etc. Visiting these sites provides a lot of information, including (a) tutorials, courses, training for students and newcomers, (b) staining protocols, consensus reports, common pitfalls, (c) free software, shareware, comments, and reviews of commercially available software, and (d) newsgroups, discussion of newcomers with experts, questions, answers, and news. Companies present in the Net will provide catalogs and data sheets together with references and useful protocols. This is an excellent way of obtaining updated information, cost-free. Commercial suppliers must therefore provide a high standard to justify their costs. An additional advantage to the Internet is that all those flow cytometry operators who are operating in the middle of nowhere and those users isolated in an institute of nonusers need no longer be alone or desperate. Once connected to the Internet, they can keep their knowledge up to date and develop new strategies for their individual needs.

V. Concluding Remarks New developments in flow cytometric can be expected in two fields: the technical branch and dye chemistry. The main technical development will be the expansion of the usable spectrum into the red/infra-red region. Excitation of appropriate dyes with low energy has many advantages: cheap and small semiconductor lasers are available in that spectral region; and due to neglectible autofluorescence of redilluminated cells, sensitivity would be automatically enhanced. The great stability of these dyes is of additional interest, though mainly in microscopy work. The only disadvantage is that fluorescence in infra-red cannot be seen in a microscope, a fact hard for pathologists to imagine. However, use of cameras/image systems will become routine, and pathologists’ will eventually be won over. An additional technical development will be a “pocket flow cytometer”, which is simple and robust, and practical for field analysis (e.g., calculating lymphoid subpopulations in space, Sams et al., 1999). Such flow cytometers will expand applications into environmental studies, such as hydrology (Yentsch 1990; Robertson et al., 1998). Other fields of biological sciences that will profit from flow cytometry are microbiology and virology. Of great interest will be the development of simpler sorting devices, like the switching systems currently available. By speeding up such sorting devices, a pocket flow cytometer will become a pocket flow sorter without the need for complicated adjustment and specially trained personnel. This will lead to “sorting for all” in the forseeable future. Some more sophisticated, commercially available instruments will be equipped with additional high-resolution data collecting systems, among them absolute counting systems (records counts per volume), light scatter in various directions

10/31/2000

05:27 PM

Cytology

280

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

(polarization dependent?) to more accurately detect differences in subpopulations, and fluorescence lifetime discrimination in combination with slit scan cytometry to resolve structure (see Section III.B.5.b). Some progress in calculating absolute numbers of dye molecules/binding sites per cell is of unquestionable interest. The main progress will come from developments in dye chemistry. There is an absolute need for better and more stable dyes with high specificity and low background, especially in the red/infra-red region. Possibly the recent development of nanocrystals (Bruchez et al., 1998), is a first step in this direction. Staining of cellular structures with beads containing embedded dye molecules is not new, but it has some mentioned disadvantages (Section II.B.3.a). Nanocrystals are much more smaller, in the neighbourhood of single molecules such as PE. They are photochemically stable, and can be tuned to a desired excitation/emission wavelength. Coupling to a protein seems to work now, so stoichiometric and quantitative staining can be performed.

Acknowledgments This work was partly supported by a Foerderungsbeitrag of the University of Innsbruck. I am grateful to all my coworkers, especially Arno Helmberg and Roswitha Gruber-Sconc, for helpful advice and Michael Knoflach for use of his thymocyte data. I am deeply indebted to Karine Keen-Traill for critical reading of the manuscript.

References Abdullah, S. M., Flath, B., and Presber, H. W. (1999). Comparison of different staining procedures for the flow cytometric analysis of U-937 cells infected with different Leishmania-species. I. Microbiol. Methods 37(2), 123–138. Agronskaia, A., Florians, A., van der Werf, K. O., et al. (1998). Photon counting device compatible with conventional flow cytometric data aquisition electronics. Cytometry 32, 255–259. Anderson, M., Tjioe, I., Lorinez, M., et al. (1996). Simultaneous fluorescence activated cell sorter analysis of two distinct transcriptional elements within a single cell using engineered green fluorescent proteins. Proc. Natl. Acad. Sci. USA 93, 8508–8511. Arndt-Jovin, D. J. (1990). Cellular differentiation. In (M. R. Melamed, P. F. Mullaney, M. L. Mendelsohn, Eds.), “Flow Cytometry and Sorting” 2nd ed., Wiley-Liss, New York. Arndt-Jovin, D. J., and Jovin, T. M. (1974). Computer-controlled multiparameter analysis and sorting of cells and particles. J. Histochem. Cytochem. 22, 622–625. Arndt-Jovin, D. J., and Jovin, T. M. (1977). Analysis and sorting of living cells according to deoxyribonucleic acid content. J. Histochem. Cytochem. 25, 585–589. Arndt-Jovin, D. J., and Jovin, T. M. (1989). Fluorescence labeling and microscopy of DNA. Meth. Cell Biol. 30, 417–448. Ashbury, C. L., Esposito, R., Farmer, C., et al. (1996). Fluorescence spectra of DNA dyes measured in a flow cytometer. Cytometry 24(3), 234–242. Atwater, S., and Corash, L. (1996). Advances in leukocyte differential and peripheral blood stem cell enumeration. Curr. Opin. Hematol. 3(1), 71–76.

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

281

Aubin, J. (1979). Autofluorescence of viable cultured mammalian cells. J. Histochem. Cytochem. 27, 36–43. Auer, G., Ono, J., Caspersson, T. O. (1983). Determination of the fraction of G0 cells in cytologic samples by means of simultaneous DNA and nuclear protein analysis. Analyt. Quant. Cytol. 5, 1–4. Bagwell, C. B. (1993). Theoretical aspects of flow cytometry data analysis. See Bauer, K. D., Duque, R. E., Shankey, T. V. Eds. (1993), 41–70. Bagwell, C. B., Mayo, S. W., Whetstone, S. D., et al. (1991). DNA histogram debris theory and compensation. Cytometry 12, 107–118. Baisch, H., Beck, H. P., Christensen, I. J., et al. (1982). A comparison of mathematical methods for the analysis of DNA histograms obtained by flow cytometry. Cell Tissue Kinet. 15, 235–249. Barber, L., Prince, H. M., Rossi, R., et al. (1999). Fluoro-gold: An alternative viability stain for multicolor flow cytometric analysis. Cytometry 36(4), 349–354. Bartholdi, M. F. (1990). Flow cytogenetics. Pathobiology 58(2), 118–128. Bass, D. A., Parce, J. W., DeChatelet L. R., et al. (1983). Flow cytometric studies of oxidative product formation by neutrophils: A graded response to membrane stimulation. J. Immunol. 130, 1910– 1917. Bauer, K. D., Bagwell, C. B., Giaretti, W., et al. (1993a). Consensus review of the clinical utility of DNA flow cytometry in colorectal cancer. Cytometry 14, 486–491. Bauer, K. D., Duque, R. E., Shankey, T. V. (Eds.) (1993b): “Clinical Flow Cytometry,” Williams and Wilkins, Baltimore, MD. Bauman, J. G., de Vries, P., Pronk, B., et al (1988). Purification of murine hemopoietic stem cells and committed progenitors by fluorescence activated cell sorting using wheat germ agglutinin and monoclonal antibodies. Acta Histochem. Suppl. 36, 241–253. Bayer, E., and Wilchek, M. (1978). The avidin-biotin complex as a tool in molecular biology. Trends Biochem. Sci. 3, N257. Beavis, A. J., and Kalejta, R. F. (1999). Simultaneous analysis of the cyan, yellow, and green fluorescent proteins by flow cytometry using single-laser excitation at 458nm. Cytometry Suppl. 37(1), 68–73. Bedner, E., Li, X., Gorczyca, M., et al. (1999). Analysis of apoptosis by laser scanning cytometry. Cytometry 35(3), 181–195. Ben-Bassat, H., Polliak, A., Mitrani-Rosenbaum, S., et al. (1977). Fluidity of membrane lipids and lateral mobility of concanavalin A receptors in the cell surface of normal lymphocytes and lymphocytes from patients with malignant lymphomas and leukemias. Cancer Res. 37, 1307– 1312. Benson, S. C., Mathies, R. A., and Glanzer, A. N. (1993a). Heterodimeric DNA-binding dyes designed for energy transfer: Stability and applications of the DNA compexes. Nucleic Acids Res. 21, 5720– 5726. Benson, H. C., Meyer, R. A., and Zaruba, M. E. (1979). Cellular autofluorescence—is it due to flavins? J. Histochem. Cytochem. 27, 44–48. Benson, S. C., Singh, P., and Glazer, A. N. (1993b). Heterodimeric DNA-binding dyes designed for energy transfer: Synthesis and spectroscopic properties. Nucleic Acids Res. 21, 5727–5735. Benz, C., Wiznitzer, I., and Lee, S. H. (1985). Flow cytometric analysis of fluorescein-conjugated estradiol (E-BSA-FITC) binding in breast cancer cell suspensions. Cytometry 6, 260–267. Bhalgat, M. K., Haugland, R. P., Pollack, J. S., et al. (1998). Green- and red-fluorescent nanospheres for the detection of cell surface receptors by flow cytometry. J. Immunol Methods 219, 57–68. Bhuyan, P. R., Loughman, B. E., Fraser, T. J., et al. (1976). Comparison of different methods of determining cell viability after exposure to cytotoxic compounds. Exp. Cell Res. 97, 275–280. Bigner, S., Bjerkvig, R., and Laerum, O. D., et al. (1987). DNA content and chromosomes in permanent cultured cell lines derived from malignant gliomas. Anal. Quant. Cytol. Histol. 9, 435–444. Billingham, R. E., Brent, L., and Medawar, P. B. (1953). Nature 172, 603. Bjerknes, R., and Bassoe, C. F. (1984). Phagocyte C3-mediated attachment and internalization: Flow cytometric studies using a fluorescence quenching technique. Blut 49(4), 315–323.

10/31/2000

05:27 PM

282

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

Blair, O. C., Carbone, R., and Sartorelli, A. C. (1985). Differentiation of HL-60 promyelocytic leukemia cells monitored by flow cytometric measurement of nitro blue tetrazolium (NBT) reduction. Cytometry 6, 54–61. Blennow, E., Telenius, H., Larsson, C., et al. (1992). Complete characterization of a large marker chromosome by reverse and forward chromosome painting. Hum. Genet. 90(4), 371–374. Boeck, G., Huber, L. A., Wick, G., et al. (1989). Use of a FACS III for fluorescence depolarization with DPH. J. Histochem. Cytochem. 37(11), 1653–1658. Boeck, G., Steinlein, P., and Huber, L. A. (1997). Cell biologists sort things out: Analysis and purification of intracellular organelles by flow cytometry. Trends Cell Biol. 7, 499–503. Boeck, G., Steinlein, P., Haberfellner, M., et al. (1998). Fluorescence-activated sorting of endocytic organelles. In (J. E. Celis, Ed.), “Cell Biology: A Laboratory Handbook” 2nd ed., Vol. 2, pp. 63–69, Academic Press, Orlando, FL. Boehmer, R. M., and King, N. J. C. (1984). Flow cytometric analysis of immunogold cell surface label. Cytometry 5, 543–546. Bogh, L. D., and Duling, T. A. (1993). Flow cytometry instrumentation in research and clinical laboratories. Clin. Lab. Sci. 6(3), 167–173. Bohn, B. (1976). High-sensitivity cytofluorometric quantitation of lectin and hormone binding to surfaces of living cells. Exp. Cell Res. 103, 39–46. Bohn, B. (1980). Flow cytometry: A novel approach for the quantitative analysis of receptor-ligand interactions on surfaces of living cells. Mol. Cell Endocrinol. 20, 1–15. Bonner, W. A., Hulett, H. R., Sweet, R. G., et al. (1972). Fluorescence activated cell sorting. Rev. Sci. Instrum. 43, 404–409. Bottiroli, G., Croce, A. C., and Ramponi, R. (1992). Fluorescence resonance energy transfer imaging as a tool for in situ evaluation of cell morphofunctional characteristics. J. Photochem. Photobiol. 12(4), 413–416. Brandt, C. A., Kierkegaard, O., Hindkjaer, J., et al. (1993). Ring chromosome 20 with loss of telomeric sequences detected by multicolor PRINS. Clin. Genet. 44(1), 26–31. Brecher, G., Schneiderman, M., and Williams, G. Z. (1956). Evaluation of electronic cell counter. Am. J. Clin. Pathol. 26, 1439. Bruchez, M., Moronne, M., Gin, P., et al. (1998). Semiconductor nanocrystals as fluorescent biological labels. Science 281, 2013–2018. Burchiel, S. W., Lauer, F. T., Gurule, D., et al. (1999). Uses and future applications of flow cytometry in immunotoxicity testing. Methods 19(1), 28–35. Burgess, S. C., and Davison, T. F. (1999). Counting absolute numbers of specific leukocyte subpopulations in avian whole blood using a single-step flow cytometric technique: comparison of two inbred lines of chickens. J. Immunol. Meth. 227(1–2), 169–76. Burow, S., and Valet, G. (1987). Flow-cytometric characterization of stimulation, free radical formation, peroxidase activity and phagozytosis of human granulocytes with 2,7-dichlorofluorescein (DCF). Eur. J. Cell Biol. 43, 128–133. Calfie, M., Tu, Y., Euskirchen, G., et al. (1994). Green fluorescent protein as a marker for gene expression. Science 263, 802–805. Cambier, J. L., Kay, D. B., and Wheeless, L. L. (1979). A multidimensional slit-scan flow system. J. Histochem. Cytochem. 27, 321–324. Casado, J. A., Merino, J., Cid, J., et al. (1993). Simultaneous evaluation of phagozytosis and Fc gamma R-mediated oxidative burst in human monocytes by a simple flow cytometry method. J. Immunol. Meth. 159, 173–176. Caspersson, T. (1950). “Cell Growth and Cell Function.” Norton, New York. Caspersson, T., and Schultz, J. (1938). Nucleic acid metabolism of the chromosomes in relation to gene reproduction. Nature 142, 294. Celis, J. E., Bravo, R., Larsen, P. M., et al. (1984). Cyclin: A nuclear protein whose level correlates directly with the proliferating state of normal as well as transformed cells. Leukemia Res. 8, 143–157.

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

283

Chan, S. S., Arndt-Jovin, D., and Jovin, T. M. (1979). Proximity of lectin receptors on the cell surface measured by fluorescence energy transfer in a flow system. J. Histochem. Cytochem. 27, 56–64. Chang, A., Hammond, T. G., Sun, T. T., et al. (1994). Permeability properties of the mammalian bladder apical membrane. Am. J. Physiol. 267, C1483–C1492. Chase, E. S., and Hoffman, R. A. (1998). Resolution of dimly fluorescent particles: A practical measure of fluorescence sensitivity. Cytometry 33, 267–279. Chatelier, R. C., and Ashcroft, R. G. (1987). Calibration of flow cytometric fluorescence standards using the isoparametric analysis of ligand binding. Cytometry 8, 632–636. Chen, L. B. (1989). Fluorescent labeling of mitochondria. Methods Cell Biol. 29, 103–123. Chu, Y., Wang, R., Schmid, I., et al. (1999). Analysis with flow cytometry of green fluorescent protein expression in leukemic cells. Cytometry 36, 333–339. Collins, J. M., Scott,R. B., McClish, D. K., et al. (1991). Altered membrane anisotropy gradients of plasma membranes of living peripheral blood leukocytes in aging and Alzheimer’s disease. Mech. Ageing Dev. 59, 153–162. Condrau, M. A., Schwendener, R. A., Niederer, P., et al. (1994a). Time-resolved flow cytometry for the measurement of lanthanide chelate fluorescence: I. Concept and theoretical evaluation. Cytometry 16(3), 187–194. Condrau, M. A., Schwendener, R. A., Zimmermann, M., et al. (1994b). Time-resolved flow cytometry for the measurement of lanthanide chelate fluorescence: II. Instrument design and experimental results. Cytometry 16(3), 195–205. Coons, A. H., Creech, H. J., and Jones, R. N. (1941). Immunological properties of an antibody containing a fluorescent group. Proc. Soc. Exp. Biol. Med. 47, 200. Coons, A. H., and Kaplan, M. H. (1950). Localization of antigen in tissue cells. II. Improvements in a method for the detection of antigen by means of fluorescent antibody. J. Exp. Med. 91, 1. Cormack, B., Valdivia, R., and Falkow, S. (1996). FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38. Corsetti, J. P., Sotirchos, S. V., Cox, C., et al. (1988). Correction of cellular autofluorescence in flow cytometry by mathematical modeling of cellular fluorescence. Cytometry 9, 539–547. Corsetti, J. P., Sparks, J. D., Sikora, B., et al. (1993). Cellular heterogeneity in binding and uptake of low-density lipoprotein in primary rat hepatocytes. Hepatology 17, 645–650. Corsetti, J. P., Weidner, C. H., Cianci, J., et al. (1991). The labeling of lipoproteins for studies of cellular binding with a fluorescent lipophilic dye. Anal. Biochem. 195, 122–128. Cossarizza, A., Baccarani-Contri, M., Kalashnikova, G., et al. (1993). A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5 , 6,6 -tetrachloro-1,1 , 3,3 -tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochem. Biophys. Res. Commun. 197(1), 40–50. Cossarizza, A., Ceccarelli, D., and Masini, A. (1996). Functional heterogeneity of an isolated mitochondrial population revealed by cytofluorometric analysis at the single organelle level. Exp. Cell Res. 222(1), 84–94. Coulter, W. H. (1956). High speed automatic blood cell counter and cell size analyzer. Proc. Natl. Electron. Conf. 12, 1034. Cram, L. S. (1990). Flow cytogenetics and chromosome sorting. Human Cell 3(2), 99–106. Crissman, H. A., Darzynkiewicz, Z., Tobey, R. A., et al. (1985). Correlated measurements of DNA, RNA, and protein in individual cells by flow cytometry. Science 228, 1321–1324. Crissman, H. A., Stevenson, A. P., Orlicky, D. J., et al. (1978). Detailed studies on the application of three fluorescent antibiotics for DNA staining in flow cytometry. Stain Technol. 53(6), 321–330. Crissman, H. A., and Steinkamp, J. A. (1973). Rapid, simultaneous measurement of DNA, protein, and cell volume in single cells from large mammalian cell populations. J. Cell Biol. 59, 766–771. Crissman, H. A., and Steinkamp, J. A. (1993). Cell cycle-related changes in chromatin structure detected by flow cytometry using multiple DNA fluorochromes. Eur. J. Histochem. 37(2), 129–138. Crissman, H. A., Tobey, R. A. (1974). Cell-cycle analysis in 20 minutes. Science 184, 1297–1298.

10/31/2000

05:27 PM

284

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

Crissman, H. A., and Tobey, R. A. (1990). Specific staining of DNA with the fluorescent antibiotics, mitramycin, chromomycin and olivomycin. Methods Cell Biol. 33, 97–103. Crissman, H. A., Van Egmond, J., Holdrinet, R. S., et al. (1981). Simplified method for DNA and protein staining of human hematopoietic cell samples. Cytometry 2, 59–62. Darzynkiewicz, Z., Traganos, F., and Melamed, M. R. (1980). New cell cycle compartments identified by multiparameter flow cytometry. Cytometry 1, 98–108. Darzynkiewicz, Z. (1993): The cell cycle: Application of flow cytometry in studies of cell reproduction. In (K. D. Bauer, R. E. Duque, and T. V. Shankey, Eds.), “Clinical Flow Cytometry,” pp. 13–39, Williams & Wilkins, Baltimore. Darzynkiewicz, Z. (1994). Simultaneous analysis of cellular RNA and DNA content. Methods Cell Biol. 41, 401–420. Darzynkiewicz, Z., Bedner, E., Traganos, F., et al. (1998). Critical aspects in the analysis of apoptosis and necrosis. Human Cell 11(1), 3–12. Darzynkiewicz, Z., Bedner, E., Li, X., Gorczyca, W., Melamed, M. R. (1999). Laser-scanning cytometry: A new instrumentation with many applications. Exp. Cell Res. 249(1), 1–12. Darzynkiewicz, Z., Bruno, S., Del Bino., et al. (1992). Features of apoptotic cells measured by flow cytometry. Cytometry 13(8), 795–808. Darzynkiewicz, Z., Kapuscinski, J., Traganos, F., et al. (1987). Applications of pyronin Y in cytochemistry of nucleic acids. Cytometry 8, 138–145. Darzynkiewicz, Z., Li, X., and Gong, J. (1994). Assays of cell viability: Discrimination of cells dying by apoptosis. Methods Cell Biol. 41, 15–38. Darzynkiewicz, Z., and Traganos, F. (1990). Multiparameter flow cytometry in studies of the cell cycle. In (M. R. Melamed, T. Lindmo, and M. L. Mendelsohn, Eds.), “Flow Cytometry and Sorting,” pp. 468–502, Wiley-Liss, New York. Darzynkiewicz, Z., Traganos, F., Kapuscinski, J., Staiano-Coico, I., and Melamed, M. R. (1984). Accessibility of DNA in situ to various fluorochromes: Relationship to chromatin changes during erythroid differentiation of Friend leukemia cells. Cytometry 5, 355–363. Demers, S. (1991). Particle analysis in oceanography. NATO Adv. Study Inst. Ser. G, Ecol. Sci. 27, 416. Demo, S. D., Masuda, E., Rossi, A. B., et al. (1999). Quantitative measurement of mast cell degranulation using a novel flow cytometric annexin-V binding assay. Cytometry 36(4), 340–348. Deng, Y., Bennink, J. R., and Kang, H. C. (1995). Fluorescent conjugates of brefeldin A selectively stain the endoplasmic reticulum and Golgi complex of living cells. J. Histochem. Cytochem. 43(9), 907–915. De Sanctis, J. B., Blanca, I., Bianco, N. E. (1995). Expression of different lipoprotein receptors in natural killer cells and their effect on natural killer proliferative and cytotoxic activity. Immunology 86(3), 399–407. Dive, C., Workman, P., Watson, J. V. (1987). Improved methodology for intracellular enzyme reaction and inhibition kinetics by flow cytometry. Cytometry 8, 552–561. Dixon, S. C., Miller, N. G. A., Carter, N. P., et al. (1992). Bivariate flow cytometry of farm animals. A potential tool for gene mapping. Anim. Genet. 23, 203–210. Dolbeare, F. (1995). Bromodeoxyuridine: A diagnostic tool in biology and medicine, Part I: Historical perspectives, histochemical methods and cell kinetics. Histochem. J. 27(5), 339–369. Dolbeare, F., Gratzner, H. G., Pallavicini, M. G., et al. (1983). Flow cytometric measurement of total DNA content and incorporated bromodeoxyuridine. Proc. Natl. Acad. Sci. USA 80, 5573–5577. Dow, J. (1998). Internet resources. In (J. E. Celis, Ed.), “Cell Biology: A Laboratory Handbook,” 2nd ed., Vol. 2, pp. 518–533, Academic Press, Orlando, FL. Dubelaar, G. B. J., Groenewegen, A. C., Stokdijk., et al. (1989). Optical plankton analyser: A flow cytometer for plankton analysis. II: Specifications. Cytometry 10, 529–539. Dubelaar, G. B. J., Visser, J. W. M., and Donze, M. (1987). Anomalous behaviour of forward and perpendicular light scattering of a cyanobacterium owing to gas vacuoles. Cytometry 8, 405– 412.

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

285

Dudeja, P. K., Anderson, K. M., and Harris, J. S. (1995). Reversal of multidrug resistance phenotype by surfactants: Relationship to membrane lipid fluidity. Arch. Biochem. Biophys. 319(1), 309– 315. Duhnen, J., Stegemann, J., Wiezorek, C., et al. (1983). A new fluid switching flow sorter. Histochemistry 77, 117–121. Duijndam, W. A., and van Duijn, P. (1988). Flow cytometric determination of carbohydrates in human erythrocytes. Histochemistry 88, 263–265. Duque, R. E. (1993). Flow cytometric analysis of lymphomas and acute leukemias. Ann. NY Acad. Sci. 677, 309–325. Duque, R. E., Andreeff, M., Braylan, R. C., et al. (1993). Consensus review of the clinical utility of DNA flow cytometry in neoplastic hematopathology. Cytometry 14, 492–496. Ehrlich, P. (1897). Klin. Jahrb. 6, 299. English transl.: (1959). In “Collected Papers of Paul Ehrlich,” 2, 107–125, Pergamon, Oxford. Ehrlich, P., Lazarus A. (1900). “Histology of the Blood,” Cambridge University Press, Cambridge. Elias, J. M. (1997). Cell proliferation indexes: A biomarker in solid tumors. Biotech. Histochem. 72(2), 78–85. Everson, D., Darzynkiewicz, Z., Jost, L., et al. (1986). Changes in accessibility of DNA to various fluorochromes during spermatogenesis. Cytometry 7, 45–53. Fantes, J. A., Green, D. K., and Cooke, H. J. (1983). Purifying human Y chromosomes by flow cytometry and sorting. Cytometry 4(1), 88–91. Fantes, J. A., Green, D. K., and Sharkey, A. (1994). Chromosome sorting by flow cytometry. Production of DNA libraries and gene mapping. Methods Mol. Biol. 29, 205–219. Ferguson-Smith, M. A. (1997). Genetic analysis by chromosome sorting and painting: Phylogenetic and diagnostic applications. Eur. J. Human Genet. 5(5), 253–265. Ferlini, C., Di Cesare, S., Rainaldi, G., et al. (1996). Flow cytometric analysis of the early phases of apoptosis by cellular and nuclear techniques. Cytometry 24(2), 106–115. Feulgen, R., and Rossenbeck, H. (1924). Mikroskopisch-chemischer Nachweis einer Nucleinsaeure vom Typus der Thymonucleinsaeure und auf die darauf beruhende elektive Faerbung von Zellkernen in mikroskopischen Praeparaten. Hoppe-Seyler’s Z. Physiol. Chem. 135, 203–214. Finkel, G. C., Grand, S., Ehrlich, M. P., et al. (1970). Cytologic screening automated by Cytoscreener. J. Assn. Adv. Med. Instrum. 4, 106. Ford, J. W., Welling, T. H. 3rd, and Stanley, J. C. (1996). PKH26 and 1251-PKH95: Characterization and efficacy as labels for in vitro and in vivo endothelial cell localization and tracking. J. Surg. Res. 62(1), 23–28. Fouchet, P., Jayat, C., Hechard, Y., et al. (1993). Recent advances of flow cytometry in fundamental and applied microbiology. Biol. Cell 78, 95–109. Fried, J., Perez, A. G., and Clarkson, B. D. (1976). Flow cytofluorometric analysis of cell cycle distributions using propidium iodide. Properties of the method and mathematical analysis of the data. J. Cell Biol. 71, 172–181. Fried, J., Perez, A. G., Clarkson, B. D. (1978). Rapid hypotonic method for flow cytofluorometry of monolayer cell cultures. Some pitfalls in staining and data analysis. J. Histochem. Cytochem. 26, 921–933. Friedman, H. P. (1950). The use of ultraviolet light and fluorescent dyes in the detection of uterine cancer by vaginal smear. Am. J. Obstet. Gynec. 59, 852. Fulwyler, M. J. (1965). Electronic separation of biological cells by volume. Science 150, 910–911. Gerson, D., Kiefer, H., and Eufe, W. (1982). Intracellular pH of mitogen-stimulated lymphocytes. Science 216, 1009. Giaretti, W., and Nusse, M. (1994). Light scatter of isolated cell nuclei as a parameter discriminating the cell-cycle subcompartments. Methods Cell Biol. 41, 389–400. Gill, J. E., Jotz, M. M., Young, S., et al. (1975). 7-Amino-actinomycin D as a cytochemical probe. I. Spectral properties. J. Histochem. Cytochem. 23, 793–799.

10/31/2000

05:27 PM

286

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

Gong, J., Li, X., Traganos, F., et al. (1994). Expression of G1 and G2 cyclins measured in individual cells by multiparameter flow cytometry: A new tool in the analysis of the cell cycle. Cell Prolif. 27, 357–371. Gong, J., Traganos, F., and Darzynkiewicz, Z. (1993). Expression of cyclins B and E in individual MOLT-4 cells and in stimulated human lymphocytes during their progression through the cell cycle. Int. J. Oncol. 3, 1037–1042. Gratama, J. W., D’Hautcourt, J. L., Mandy, F., et al. (1998). Flow cytometric quantitation of immunofluorescence intensity: Problems and perspectives. Cytometry 33, 166–178. Gratzner, H. G. (1982). Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: A new reagent for detection of DNA replication. Science 218, 474–475. Gratzner, H. G., Leif, R. C., Ingram, D. J., et al. (1975). The use of antibody specific for bromodeoxyuridine for the immunofluorescent determination of DNA replication in single cells and chromosomes. Exp. Cell Res. 95, 88–94. Gray, D. W., Gohde, W., Carter, N., et al. (1989). Separation of pancreatic islets by fluorescenceactivated sorting. Diabetes 38, 133–135. Gray, J. W. (Ed.) (1989). “Flow Cytogenetics” Academic Press. New York. Gray, J. W., Carrano, A. V., Steinmetz, L. L., et al. (1975). Chromosome measurement and sorting by flow systems. Proc. Natl. Acad. Sci. USA 72, 1231–1234. Gray, J. W., and Darzynkiewicz, Z. (Eds.) (1987): “Techniques in Cell Cycle Analysis.” Humana Press, Clifton. Gray, J. W., Dean, P. N., Fuscoe, J. C., et al. (1987). High-speed chromosome sorting. Science 238, 323–329. Gray, J. W., Dolbeare, F., and Pallavicine, M. G. (1990). Quantitative cell cycle analysis. In (M. R. Melamed, T. Lindmo, and M. L. Mendelsohn, Eds.) “Flow Cytometry and Sorting,” 2nd. ed., pp. 445–467, Wiley-Liss, New York. Gray, J. W., Kuo, W. L., and Pinkel, D. (1991). Molecular cytometry applied to detection and characterization of disease-linked chromosome aberrations. Baillieres Clin. Haematol. 4(3), 683– 693. Green, D. K. (1990). Analysing and sorting human chromosomes. J. Microsc. 159, 237–244. Gross, H. J., Verwer, B., Houck, D., et al. (1993). Detection of rare cells at a frequency of one per million by flow cytometry. Cytometry 14(5), 519–526. Gross, H. J., Verwer, B., and Houck, D., et al. (1995). Model study detecting breast cancer cells in peripheral blood mononuclear cells at frequencies as low as 107. Proc. Natl. Acad. Sci. USA 92(2), 537–541. Grunwald, D. (1993). Flow cytometry and RNA studies. Biol. Cell 78, 27–30. Grynkiwicz, G., Poenie, M., and Tsien, R. Y. (1985). A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450. Guasch, R. M., Guerri, C., and O’Connor, J. E. (1993). Flow cytometric analysis of concanavalin A binding to isolated Golgi fractions from rat liver. Exp. Cell Res. 207(1), 136–141. Guasch, R. M., Guerri, C., and O’Connor, J. E. (1995). Study of surface carbohydrates on isolated Golgi subfractions by fluorescent-lectin binding and flow cytometry. Cytometry 19(2), 112–118. Guindulain, T., Comas, J., and Vives-Rego, J. (1997). Use of nucleic acid dyes SYTO-13, TOTO-1, and YOYO-1 in the study of Escherichia coli and marine prokariotic populations by flow cytometry. Appl. Environ. Microbiol. 63(11), 4608–4611. Hamori, E., Arndt-Jovin, D. J., Grimwade, B. G., et al. (1980). Selection of viable cells with known DNA content. Cytometry 1, 13205. Harel-Bellan, A., Krief, P., and Rimsky, L., et al. (1990). Flow cytometry resonance energy transfer suggests an association between low-affinity interleukin 2 binding sites and HLA class I molecules. Biochem. J. 268, 35–40. Hassall, D. G. (1992). Three probe flow cytometry of a human foam-cell forming macrophage. Cytometry 13(4), 381–388.

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

287

Hassall, D. G., and Graham, A. (1995). Changes in free cholesterol content, measured by filipin fluorescence and flow cytometry, correlate with changes in cholesterol biosynthesis in THP-1 macrophages. Cytometry 21(4), 352–362. Haugland, R. P. (1999). “Handbook of Fluorescent Probes and Research Chemicals,” 7th ed., Molecular Probes, Eugene, OR. Havenith, C. E., Breedijk, A. J., van Miert, P. P., et al. (1993). Separation of alveolar macrophages and dentritic cells via autofluorescence: Phenotypical and functional characterization. J. Leukoc. Biol. 53, 504–510. Hedley, D. W., Clark, G. M., Cornelisse, C. J., et al. (1993). Consensus review of the clinical utility of DNA cytometry in carcinoma of the breast. Report of the DNA Cytometry Consensus Conference. Cytometry 14, 482–485. Hedley, D. W., and Chow, S. (1994). Evaluation of methods for measuring cellular glutathione content using flow cytometry. Cytometry 15, 349–358. Hedley, D. W., Friedlander, M. L., Taylor, I. W., et al. (1983). Method for analysis of cellular DNA content of paraffin-embedded pathological material using flow cytometry. J. Histochem. Cytochem. 31, 1333–1335. Hedley, D. W., Friedlander, M. L., Taylor, I. W., et al. (1984). DNA flow cytometry of paraffin-embedded tissues. Cytometry 5, 660–666. Hedley, D. W., Friedlander, M. L., and Taylor, I. W. (1985). Application of DNA flow cytometry to paraffin-embedded archival material for the study of aneupoidy and its clinical significance. Cytometry 6, 327–333. Hedley, D. W., Shankey, T. V., and Wheeless, L. L. (1993). DNA cytometry consensus conference. Cytometry 14, 471. Helm, R., Cubitt, A., and Talen, R. (1995). Improved green fluorescence protein. Nature 373, 663–664. Helmberg, A., Boeck, G., Wolf, H., et al. (1989). An orally administered bacterial immunomodulator primes rabbit neutrophils for increased oxidative burst in response to opsonized zymosan. Infect. Immun. 57(11), 3576–3580. Henderson, L. O., Marti, G. E., Gaigalas, A., et al. (1998). Terminology and nomenclature for standardization in quantitative fluorescence cytometry. Cytometry 33, 97–105. Hercher, M., Mueller, W., and Shapiro, H. M. (1979). Detection and discrimination of individual viruses by flow cytometry. J. Histochem. Cytochem. 27, 350–352. Hiddemann, W., Schumann, J., Andreeff, M., et al. (1984). Convention on nomenclature for DNA cytometry. Cancer Genet. Cytogenet. 13(2), 181–183. Hirons, G. T., Fawcett, J. J., and Crissman, H. A. (1994). TOTO and YOYO: New very bright fluorochromes for DNA content analyses by flow cytometry. Cytometry 15(2), 129–140. Hirschfeld, T. (1976). Optical microscopic observation of single small molecules. Appl. Opt. 15, 2965. Hoffman, J. F., and Laris, P. C. (1974). Determination of membrane potentials in human and Amphiuma red cells by means of a fluorescent probe. J. Physiol. 239, 519–552. Hopwood, D. (1985). Cell and tissue fixation, 1972–1982. Histochem. J. 17, 389–442. Horan, P. K., Melnicoff, M. J., Jensen, B. D., et al. (1990). Fluorescent cell labeling for in vivo and in vitro cell tracking. Methods Cell Biol. 33, 469–490. Horan, P. K., and Wheeless, L. L. (1977). Quantitative single cell analysis and sorting. Science 189, 149–157. Horobin, R. W. (1969). The impurities of biological dyes: Their detection, removal, occurrence and histological significance—A review. Histochem. J. 1, 231. Hossain, A. M., Barik, S., Rizk, B., et al. (1998). Prconceptional sex selection: Past, present, and future. Arch. Androl. 40(1), 3–14. Huber, L. A., Boeck, G., Juergens, G., et al. (1990). Increased expression of high-affinity low-density lipoprotein receptors on human T-blasts. Int. Arch. Allergy Appl. Immunol. 93, 205–211. Hulett, H. R., Bonner, W. A., Barrett, J., et al. (1969). Automated separation of mammalian cells as a function of intracellular fluorescence. Science 16, 747–749.

10/31/2000

05:27 PM

288

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

Hultin, L. E., Matud, J. L., and Giorgi, J. V. (1998). Quantitation of CD38 activation antigen expression on CD8+ T cells in HIV-1 infection using CD4 expression on CD4+ T lymphocytes as a biological calibrator. Cytometry 33(2), 123–132. Hussey, S., Yates, C. M., and Fink, G. (1986). Fluorescence activated cell sorting (FACS) as a separation method for neurofibrillary tangles in Alzheimer’s disease. J. Neurosci. Methods 16(1), 1–8. Illsley, N. P., and Verkman, A. S. (1987). Membrane chloride transport measured using a chloridesensitive fluorescent probe. Biochemistry 26, 1215–1219. Jacobberger, J. W., Fogleman, D., Lehman, J. M. (1986). Analysis of intracellular antigens by flow cytometry. Cytometry 7, 356–364. Johnson, L. A. (1995). Sex preselection by flow cytometric separation of X and Y chromosome-bearing sperm based on DNA differences: A review. Reprod. Fertil. Dev. 7(4), 893–903. Johnson, L. A. (1997). Advances in gender preselection in swine. J. Reprod. Fertil. Suppl. 52, 255–266. Johnston, J. S., Bennett, M. D., Rayburn, A. L., et al. (1999). Reference standards for determination of DNA content of plant nuclei. Am. J. Bot. 86(5), 609. Jongkind, J. F., and Verkerk, A. (1984). Cell sorting and microchemistry of cultured human fibroblasts: applications in genetics and aging research. Cytometry 5, 182–187. Jongkind, J. F., Verkerk, A., Visser, W. J., et al (1982). Isolation of autofluorescent ‘aged’ human fibroblasts by flow sorting. Exp. Cell Res. 138, 409–417. Juan, G., and Darzynkiewicz, Z. (1998). Cell cycle analysis by flow and laser scanning cytometry. In (J. E. Celis, Ed.), “Cell Biology: A Laboratory Handbook,” 2nd ed., Vol. 2, pp. 63–69, Academic Press, Orlando, FL. June, C. H., and Rabinovitch, P. S. (1994). Intracellular ionized calcium. Meth. Cell Biol. 41, 149–174. Kao, J. P. Y. (1994). Practical aspects of measuring Ca2+ with fluorescent indicators. Methods Cell Biol. 40, 155–181. Kachel, V., Glossner, E., and Schneider, H. (1982). A new flow cytometric transducer for fast sample throughput and time resolved kinetic studies of biological cells and other particles. Cytometry 3, 202–212. Kalejta, R. F., Shenk, T., and Beavis, A. J. (1997). Use of a membrane-localized green fluorescent protein allows simultaneous identification of transfected cells and cell cycle analysis by flow cytometry. Cytometry 29, 286–291. Kamentsky, L. A., and Melamed, I. R. (1969). Instrumentation for automated examinations of cellular specimens. Proc. IEEE 57, 2007. Kamentsky, L. A., Melamed, M. R., and Derman, H. (1965). Spectrophotometer: New instrument for ultrarapid cell analysis. Science 150, 630–631. Kapuscinski, J. (1995). DAPI: A DNA-specific fluorescent probe. Biotech. Histochem. 70(5), 220–233. Kelley, K. A. (1989). Sample station modification providing on-line reagent addition and reduced sample transit time for flow cytometers. Cytometry 10, 796–800. Kelley, K. A. (1991). Very early detection of changes associated with cellular activation using a modified flow cytometer. Cytometry 12, 464–468. Keren, D. F. (Ed.) 1989): “Flow Cytometry in Clinical Diagnosis.” ASCP Press, Chicago. Kerker, M., Chew, H., McNulty, P. J., et al. (1979). Light scattering and fluorescence by small particles having internal structure. J. Histochem. Cytochem. 27, 250–263. Kerker, M., Van Dilla, M. A., Brunsting, A., et al. (1982). Is the central dogma of flow cytometry true: that fluorescence intensity is proportional to cellular dye content?. Cytometry 3, 71–78. Koehler, G., and Milstein, C. (1975). Nature. 256, 495. Koenig, S. H., Brown, R. D., Kamentsky, L. A., et al. (1968). Efficacy of a rapid cell spectrophotometer in screening for cervical cancer. Cancer 21, 1019. Kosenow, W. (1952). Die Fluorochromierung mit Acridinorange, eine Methode zur Lebendbeobachtung gefaerbter Blutzellen. Acta Haemat. 7, 217. Krause, D., Shearman, C., and Lang, W., et al. (1990). Determination of affinities of murine and chimeric anti alpha/beta-T-cell receptor antibodies by flow cytometry. Behring Inst. Mitt. 87, 56–67.

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

289

Kraemer, P. M., Tobey, R. A., and VanDilla, M. A. (1973). Flow microfluorimetric studies of lectin binding to mammalian cells, I. General features. J. Cell Physiol. 81, 305–314. Krishan, A. (1975). Rapid flow cytometric analysis of mammalian cell cycle by propidium iodide staining. J. Cell Biol. 66, 188–193. Ktistakis, N. T., Kao, C. Y., Wang, R. H., et al. (1995). A fluorescent lipid analogue can be used to monitor secretory activity and for isolation of mammalian secretion mutants. Mol. Biol. Cell 6(2), 135–150. Kurki, P., Vanderlaan, M., Dolbeare, F., et al. (1986). Expression of proliferating cell nuclear antigen (PCNA)/cyclin during the cell cycle. Exp. Cell Res. 166, 209–219. Lacombe, F., and Belloc, F. (1996). Flow cytometry study of cell cycle, apoptosis and drug resistance in acute leukemia. Hematol. Cell Ther. 38(6), 495–504. Laerum, O. D., Bjerknes, R. 1992): “Flow Cytometry in Hematology.” Academic Press, London. Lalande, M. E., and Miller, R. G. (1979). Fluorescence flow analysis of lymphocyte activation using Hoechst 33342 dye. J. Histochem. Cytochem. 27, 394–397. Landay, A. L., and Muirhead, K. A. (1989). Procedural guidelines for performing immunophenotyping by flow cytometry. Clin. Immunol. Immunopathol. 52(1), 48–60. Landsteiner, K. (1899). Zentralbl. Bakteriol. 16, 13. Lane, T. A., and Chen, T. T. (1991). Heterologous down-modulation of luteinizing hormone receptors by prolactin: A flow cytometry study. Endocrinology 128(4), 1833–1840. Latt, S. A., Marino, M., Lalande, M. (1984). New fluorochromes, compatible with high wavelength excitation, for flow cytometric analysis of cellular nucleic acids. Cytometry 5, 339–347. Latt, S. A., and Stetten, G. (1976). Spectral studies on 33258 Hoechst and related bisbenzimidazole dyes useful for fluorescent detection of deoxyribonucleic acid synthesis. J. Histochem. Cytochem. 24, 24–33. Lazzari, K. G., Proto, P. J., and Simons, E. R. (1986). Simultaneous measurement of stimulus-induced changes in cytoplasmic Ca2+ and in membrane potential of human neutrophils. J. Biol. Chem. 261, 9710–9713. Leary, J. F., Schmidt, D. F., Gram, J. G., et al. (1994). High-speed flow cytometric analysis and sorting of human fetal cells from maternal blood for molecular characterization. Ann. NY Acad. Sci. 731, 138–141. Leary, J. F., Todd, P., Wood, J. C. S., et al. (1979). Laser flow cytometric light scatter and fluorescence puls width and pulse risetime sizing of mammalian cells. J. Histochem. Cytochem. 27, 315–320. Lebaron, P., Parthuisot, N., and Catala, P. (1998). Comparison of blue nucleic acid dyes for flow cytometric enumeration of bacteria in aquatic systems. Appl. Environ Microbiol 64(5), 1725– 1730. Lewis, D. E., Schober, W., Murrell, S., et al. (1996). Rare event selection of fetal nucleated erythrocytes in maternal blood by flow cytometry. Cytometry 23(3), 218–227. Liang, B., and Petty, H. R. (1992). Imaging neutrophil activation: Analysis of the translocation and utilization of NAD(P)H-associated autofluorescence during antibody-dependent target oxidation. J. Cell Physiol. 152, 145–156. Liu, H. S., Jan, M. S., Chou, C. K., et al. (1999). Is green fluorescent protein toxic to the living cells? Biochem. Biophys. Res. Commun. 260(3), 712–717. Liu, Z., Bushnell, W. R., and Brambl, R. (1987). Potentiometric cyanine dyes are sensitive probes for mitochondria in intact plant cells. Plant Physiol. 84, 1385. Lloyd, D. (1993). “Flow Cytometry in Microbiology,” pp. 188, Springer, London. Loken, M. R., and Herzenberg, L. A. (1975). Analysis of cell populations with a fluorescence-activated cell sorter. Ann. NY Acad. Sci. 254, 163. Loken, M. R. (1980a). Simultaneous quantitation of Hoechst 33342 and immunofluorescence on viable cells using a fluorescence activated cell sorter. Cytometry 1, 136–142. Loken, M. R. (1980b). Separation of viable T and B lymphocytes using a cytochemical stain, Hoechst 33342. J. Histochem. Cytochem. 28, 36–39.

10/31/2000

05:27 PM

290

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

Loken, M. R., Brosnan, J. M., Bach, B. A., et al. (1990). Establishing optimal lymphocyte gates for immunophenotyping by flow cytometry. Cytometry 11, 453–459. Loo, D. T., and Rillema, J. R. (1998). Measurement of cell death. Methods Cell Biol. 57, 251–264. Look, A. T. (1988). The cytogenetics of childhood leukemia: Clinical and biologic implications. Pediatr. Clin. North Am. 35(4), 723–741. Lyons, A. B. (1999). Divided we stand: Tracking cell proliferation with carboxyfluorescein diacetate succinimidyl ester. Immunol. Cell Biol. 77(6), 509–515. Macey, M. G. (1994). “Flow Cytometry: Clinical Applications.” Blackwell Scientific, Oxford. Maczek, C., Recheis, H., Boeck, G., et al. (1996). Comparison of low density lipoprotein uptake by different human lymphocyte subsets: A new method using double-fluorescence staining. J. Lipid Res. 37(6), 1363–1371. Maftah, A., Petit, J. M., Ratinaud, M. H., et al. (1989). 10-N nonyl-acridine orange: A fluorescent probe which stains mitochondria independently of their energetic state. Biochem. Biophys. Res. Commun. 164(1), 185–190. Mancini, M., Sedghinasab, M., Knowlton, K., et al. (1998). Flow cytometric measurement of mitochondrial mass and function: A novel method for assessing chemoresistance. Ann. Surg. Oncol. 5(3), 287–295. Marie, D., Vaulot, D., and Partensky, F. (1996). Application of the novel nucleic acid dyes YOYO1, YO-PRO-1, and PicoGreen for flow cytometric analysis of marine prokaryotes. Appl. Environ. Microbiol. 62(5), 1649–1655. Mason, D. J., Shanmuganathan, S., Mortimer, F. C., et al. (1998). A fluorescent Gram stain for flow cytometry and epifluorescence microscopy. Appl. Environ. Microbiol. 64(7), 2681–2685. Matko, J., and Edidin, M. (1997). Energy transfer methods for detecting molecular clusters on cell surfaces. Methods Enzymol. 278, 444–462. Mattern, C. F. T., Brackett, F. S., and Olson, B. J. (1957). Determination of number and size of particles by electrical gating: Blood cells. J. Appl. Physiol. 10, 56. Matyus, L. (1992). Fluorescence resonance energy transfer measurements on cell surfaces. A spectroscopic tool for determining protein interactions. J. Photochem. Photobiol. 12(4), 323–337. Matzdorff, A. C., Berchner, D., Kuhnel, G., Kemkes-Matthes, B., Pralle, H., and Voss, R. (1998). Relative and absolute changes of activated platelets, microparticles and platelet aggregates after activation in vitro. Haemostasis 28(6), 277–288. Mayall, B. H. (1993). Quality control and quality assurance in flow cytometry. Ann. NY Acad. Sci. 677, 78–81. McCoy, J. P., and Keren, D. F. (1999). Current practices in clinical flow cytometry. A practice survey by the American Society of Clinical Pathologists. Am. J. Clin. Pathol. 111(2), 161–168. McGann, L. E., Walterson, M. L., and Hogg, L. M. (1988). Light scattering and cell volumes in osmotically stressed and frozen-thawed cells. Cytometry 9, 33–38. Melamed, M. R., Mullaney, P. F., Mendelsohn, M. L. (1990). “Flow Cytometry and Sorting,” 2nd ed., Wiley-Liss, New York. Mellors, R. C. (1958). “Analytical Cytology.” McGraw-Hill, New York. Mellors, R. C., Keane, J. F., and Papanicolaou, G. N. (1952). Nucleic acid content of the squamous cancer cell. Science 116, 265. Mellors, R. C., and Silver, R. (1951). A microfluorometric scanner for the differential detection of cells: Application to exfoliative cytology. Science 114, 356. Metezeau, P., Schmitz, A., and Frelat, G. (1993). Analysis and sorting of chromosomes by flow cytometry: New trends. Biol. Cell 78, 31–39. Meuwis, K., Boens, N., De Schryver, F. C., et al. (1995). Photophysics of the Fluorescent K+ Indicator PBFI. Biophys. J. 68, 2469–2473. Miner, R. W., and Kopac, M. J. (1956). Cancer cytology and cytochemistry. Ann. NY Acad. Sci. 63, 1033. Minta, A., Kao, J. P., and Tsien, R. Y. (1989). Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J. Biol. Chem. 264, 8171–8178.

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

291

Minta, A., and Tsien, R. Y. (1989). Fluorescent indicators for cytosolic sodium. J. Biol. Chem. 264, 19449–19457. Moerner, W. E., Peterman, E. J. G., Brasselet, S., et al. (1999). Optical methods for exploring dynamics of single copies of green fluorescent protein. Cytometry 36, 232–238. Mohler, J. L., Partin, A. W., Coffey, D. S. (1987). Correlation of prognosis to nuclear roundness and to flow cytometric light scatter. Anal. Quant. Cytol. Histol. 9(2), 156–164. Monroe, J. G., Havran, W. L., and Cambier, J. C. (1982). Enrichment of viable lymphocytes in defined cell cycle phases by sorting on the basis of pulse width of axial light extinction. Cytometry 3, 24–27. Moore, A., Donahue, C. J., Bauer, K. D., et al. (1998). Simultaneous measurement of cell cycle and apoptotic cell death. Methods Cell Biol. 57, 265–278. Morgan, G. J., and Pratt, G. (1998). Modern molecular diagnostics and the management of haematological malignancies. Clin. Lab. Haematol. 20(3), 135–141. Mueller, M. R., Lennartz, K., Nowrousian, M. R., et al. (1994). Improved flow-cytometric detection of low P-glycoprotein expression in leukaemic blasts by histogram analysis. Cytometry 15, 64– 72. Muller, C. P., Stephany, D. A., Winkler, D. F., et al. (1984). Filipin as a flow microfluorometry probe for cellular cholesterol. Cytometry 5(1), 42–54. Murphy, R. F. (1985). Analysis and isolation of endocytic vesicles by flow cytometry and sorting: Demonstration of three kinetically distinct compartments involved in fluid-phase endocytosis. Proc. Natl. Acad. Sci. USA 82(24), 8523–8526. Musgrove, E., Rugg, C., and Hedley, D. (1986). Flow cytometric measurement of cytoplasmic pH: A critical evaluation of available fluorochromes. Cytometry 7, 347–355. Nguyen, D. C., Keller, R. A., Jett, J. H., et al. (1987). Detection of single molecules of phycoerythrin in hydrodynamically focused flows by laser-induced fluorescence. Anal. Chem. 59, 2158. Nicholson, J. K. (1994). Quality control in immunophenotyping: U.S. efforts to establish common methodology and their impact. American Society for Histocompatibility and Immunogenetics. Eur. J. Histochem. 38 Suppl. 1, 7–12. Nicholson, J. K. A., and Stetler-Stevenson, M. (1998). Quantitative fluorescence: To count or not to count. Is that the question? Cytometry 34, 203–204. Nicoletti, I., Migliorati, G., Pagliacci, M. C., et al. (1991). A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods 139(2), 271–279. Nizetic, D., Zehetner, G., Monaco, A. P., et al. (1991). Construction, arraying, and high-density screening of large insert libraries of human chromosomes X and 21: Their potential use as reference libraries. Proc. Natl. Acad. Sci. USA 88(8), 3233–3237. O’Brien, M. C., and Bolton, W. E. (1995). Comparison of cell viability probes compatible with the fixation and permeabilization for combined surface and intracellular staining in flow cytometry. Cytometry 19(3), 243–255. O’Connor, J. E., Kimler, B. F., Morgan, M. C., et al. (1988). A flow cytometric assay for intracellular nonprotein thiols using mercury orange. Cytometry 9, 529–532. Oda, T., and Maeda, H. (1986). A new simple fluorometric assay for phagocytosis. J. Immunol. Meth. 88, 175–183. Omann, G. M., Coppersmith, W., Finney, D. A., et al. (1985). A convenient on-line device for reagent addition, sample mixing, and temperature control of cell suspensions in flow cytometry. Cytometry 6, 69–73. Orfao, A., Schmitz, G., Brando, B., et al. (1999). Clinically useful information provided by the flow cytometric immunophenotyping of hematological malignancies: Current status and future directions. Clin. Chem. 45(10), 1708–1717. Ormerod, M. G. (2000). “Flow-Cytometry—A Practical Approach.” 3rd ed., Oxford Press. Ormerod, M. G. (1997). Analysis of cell proliferation using the bromodeoxyuridine/Hoechst-ethidium bromide method. Methods Mol. Biol. 75, 357–365.

10/31/2000

05:27 PM

292

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

Ormerod, M. G. (1998). The study of apoptotic cells by flow cytometry. Leukemia 12(7), 1013–1025. Ost, V., Neukammer, J., and Rinneberg, H. (1998). Flow cytometric differentiation of erythrocytes and leukocytes in dilute whole blood by light scattering. Cytometry 32, 191–197. Otto, F., and Tsou, K. C. (1985). A comparative study of DAPI, DIPI, and Hoechst 33258 and 33342 as chromosomal DNA stains. Stain Technol. 60, 7–11. Otto, F. J. (1994). High-resolution analysis of nuclear DNA employing the fluorochrome DAPI. Methods Cell Biol. 41, 211–217. Pagano, R. E., and Martin, O. C. (1998). Use of fluorescent analogs of ceramide to study the Golgi apparatus of animal cells. In (J. E. Celis, Ed.), “Cell Biology: A Laboratory Handbook,” 2nd ed., Vol. 2. pp. 507–512. Academic Press, Orlando, FL. Pagano, R. E., Martin, O. C., Kang, H. C., Haugland, R. P. (1991). A novel fluorescent ceramide analogue for studying membrane traffic in animal cells: Accumulation at the Golgi apparatus results in altered spectral properties of the sphingolipid precursor. Cell Biol. 113(6), 1267–1279. Papanicolaou, G. N., and Traut, H. F. (1941). The diagnostic value of vaginal smears in carcinoma of the uterus. Am. J. Obstet. Gynec. 42, 193. Parish, C. R. (1999). Fluorescent dyes for lymphocyte migration and proliferation studies. Immunol. Cell Biol. 77(6), 499–508. Parker, J. W., Adelsberg, B., Azen, S. P., et al. (1990). Leukocyte immunophenotyping by flow cytometry in a multisite study: Standardization, quality control, and normal values in the Transfusion Safety Study. Clin. Immunol. Immunopathol. 55(2), 187–220. Peeters, J. C., Dubelaar, G. B., Ringelberg, J., et al. (1989). Optical plankton analyser: A flow cytometer for plankton analysis. I: Design considerations. Cytometry 10, 522–528. Perticarari, S., Presani, G., Mangiarotti, M. A., et al. (1991). Simultaneous flow cytometric method to measure phagocytosis and oxidative products by neutrophils. Cytometry 12, 687–693. Peters, D. (1979). A comparison of mercury arc lamp and laser illumination for flow cytometers. J. Histochem. Cytochem. 27, 241–245. Peters, D., Branscomb, E., Dean, P., et al. (1985). The LLNL high-speed sorter: Design features, operational characteristics, and biological utility. Cytometry 6, 290–301. Petersen, S. E., and Friedrich, U. (1986). A comparison between flow cytometric ploidy investigation and chromosome analysis of 32 human colorectal tumors. Cytometry 7, 307–312. Petit, P. X., Lecoeur, H., Zorn, E., et al. (1995). Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis. J. Cell Biol. 130(1), 157–167. Pinsky, B. G., Ladasky, J. J., Lakowicz, J. R., et al. (1993). Phase-resolved fluorescence lifetime measurements for flow cytometry. Cytometry 14, 123–135. Plasek, J., and Sigler, K. (1996). Slow fluorescent indicators of membrane potential: A survey of different approaches to probe response analysis. J. Photochem. Photobiol. 33(2), 101–124. Poot, M. (1998). Staining of mitochondria. In (J. E. Celis, Ed.), “Cell Biology: A Laboratory Handbook,” 2nd ed., Vol. 2, pp. 513–517. Academic Press, Orlando, FL. Poot, M., Gibson, L. L., and Singer, V. L. (1997). Detection of apoptosis in live cells by MitoTracker red CMXRos and SYTO dye flow cytometry. Cytometry 27(4), 358–364. Poot, M., Hoehn, H., Kubbies, M., et al. (1994). Cell-cycle analysis using continuous bromodeoxyuridine labeling and Hoechst 33358-ethidium bromide bivariate flow cytometry. Methods Cell Biol. 41, 327–340. Price, B. J., Kollman, V. H., and Salzman, G. C. (1978). Light-scatter analysis of microalgae. Correlation of scatter patterns from pure and mixed asynchronous cultures. Biophys. J. 22, 29–36. Qian, J., Jenkins, R. B., and Bostwick, D. G. (1996). Potential markers of aggressiveness in prostatic intraepithelial neoplasia detected by fluorescence in situ hybridization. Eur. Urol. 30(2), 177– 184. Rabinovitch, P. S. (1993). Practical considerations for DNA content and cell cycle analysis. In (K. D. Bauer, R. E. Duque, and T. V. Shankey, Eds.), “Clinical Flow Cytometry,” pp. 117–142. Williams & Wilkins, Baltimore.

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

293

Rabinovitch, P. S., June, C. H., Grossmann, A., et al. (1986). Heterogeneity among T cells in intracellular free calcium response after mitogen stimulation with PHA or a-CD3. Simultaneous use of indo-1 and immunofluorescence with flow cytometry. J. Immunol. 137, 952–961. Rabinovitch, P. S., Torres, R. M., and Engel, D. (1986). Simultaneous cell cycle analysis and two-color surface immunofluorescence using 7-amino-actinomycin D and single laser excitation: Applications to study of cell activation and the cell cycle of murine Ly-1 B cells. J. Immunol. 136, 2769– 2775. Rabinowitz, R., Granot, E., Deckelbaum, R., et al. (1992). Antigenic differences between subsets of peripheral blood lymphocytes differing in their right angle light scatter in flow cytometric analysis. Int. Arch. Allergy Immunol. 97, 200–204. Rao, K. M., Padmanabhan, J., Kilby, D. L., et al. (1992). Flow cytometric analysis of nitric oxide production in human neutrophils using dichlorofluorescein diacetate in the presence of a calmodulin inhibitor. J. Leukoc. Biol. 51, 496–500. Reers, M., Smith, T. W., and Chen, L. B. (1991). J-aggregate formation of a carbocyanine as a fluorescent indicator of membrane potential. Biochemistry 30, 4480–4486. Rehse, M. A., Corpuz, S., Heimfeld, S., et al. (1995). Use of fluorescence threshold triggering and high-speed flow cytometry for rare event detection. Cytometry 22(4), 317–322. Rens, W., Van Oven, C. H., Stap, J., et al. (1994). Slit-scanning technique using standard cell sorter instruments for analyzing and sorting nonacrocentric human chromosomes, including small ones. Cytometry 16, 80–87. Rice, G. C., Bump, E. A., Shrieve, D. C., et al. (1986). Quantitative analysis of cellular glutathione by flow cytometry utilizing monochlorobimane: Some applications to radiation and drug resistance in vitro and in vivo. Cancer Res. 46, 6105–6110. Riedy, M. C., Muirhead, K. A., Jensen, C. P., et al. (1991). Use of a photolabeling technique to identify nonviable cells in fixed homologous of heterologous cell populations. Cytometry 12, 133–139. Robertson, B. R., Button, D. K., and Koch, A. L. (1998). Determination of the biomasses of small bacteria at low concentrations in a mixture of species with forward light scatter measurements by flow cytometry. Appl. Environ. Microbiol. 64(10), 3900–3909. Robinson, J. P. (1993). “Handbook of Flow Cytometry Methods,” Wiley-Liss, New York. Roederer, M., De Rosa, S., Gerstein, R., et al. (1997). 8 Color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity. Cytometry 29, 328–339. Roederer, M., Staal, F. G. T., Osada, H., et al. (1991). CD4 and CD8 T cells with high intracelluar glutathione level are selectively lost as the HIV infection progresses. Int. Immunol. 3, 933– 937. Roper, P. R., and Drewinko, B. (1976). Comparison of in vitro methods to determine drug-induced cell lethality. Cancer Res. 36, 2182–2188. Ropp, J. D., Donahue, C. J., Wolfgang-Kimball, D., et al. (1995). Aequores green fluorescent protein analysis by flow cytometry. Cytometry 21, 309–317. Rosenblatt, J. I., Hokanson, J. A., McLaughlin, S. R., Leary, J. F. (1997). Theoretical basis for sampling statistics useful for detecting and isolating rare cells using flow cytometry and cell sorting. Cytometry 27(3), 233–238. Roslaniec, M. C., Bell-Prince, C. S., Crissman, H. A., et al. (1997). New flow cytometric technologies for the 21st century. Human Cell 10(1), 3–10. Rothe, G., and Valet, G. (1990). Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2 ,7 -dichlorofluorescin. J. Leukoc. Biol. 47, 440–448. Roti Roti, J., Higashikubo, R., Blair, O. C., et al. (1982). Cell cycle position and nuclear protein content. Cytometry 3, 91–96. Rotman, B., and Papermaster, B. W. (1966). Membrane properties of living cells as studied by enzymatic hydrolysis of fluorogenic esters. Proc. Natl. Acad. Sci. USA 55, 134. Russell, W. C., Newman, C., and Williamson, D. H. (1975). A simple cytochemical technique for demonstration of DNA in cells infected with mycoplasmas and viruses. Nature 253, 461–462.

10/31/2000

05:27 PM

294

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

Sahlin, S., Hed, J., and Rundquist, I. (1983). Differentiation between attached and ingested immune complexes by a fluorescence quenching cytofluorometric assay. J. Immunol. Methods 60, 115–124. Salzman, G. C., Crowell, J. M., Goad, C. A., et al. (1975). A flow-system multiangle light-scattering instrument for cell characterization. Clin. Chem. 21, 1297–1304. Salzman, G. C., Crowell, J. M., Martin, J. C., et al. (1975). Cell identification by laser light scattering: Identification and separation of unstained leucocytes. Acta Cytol 19, 374–377. Sams, C. E., Crucian, B. E., Clift, V. L., et al. (1999). Development of a whole blood staining device for use during space shuttle flights. Cytometry 37, 74–80. Sasaki, D. T., Tichenor, E. H., Lopez, F., et al. (1995). Development of a clinically applicable high-speed flow cytometer for the isolation of transplantable human hematopoietic stem cells. J. Hematother. 4(6), 503–514. Saunders, A. M., and Hulett, H. R. (1969). Microfluorometry: A comparison of single measurements to a rapid flow system. J. Histochem. Cytochem. 17, 188. Schafer, I. A., Jamieson, A. M., Petrelli, M., et al. (1979). Multiangle light scattering flow cytometry of cultured human fibroblasts: Comparison of normal cells with a mutant line containing cytoplasmic inclusions. J. Histochem. Cytochem. 27, 359–365. Scheenen, W. J. J, Hofer, A. M., and Pozzan, T. (1998). Intracellular measurement of calcium using fluorescent probes. In (J.E. Celis, Ed.), “Cell Biology: A Laboratory Handbook.” 2nd Ed., Vol. 3, pp. 363–374. Academic Press, Orlando, FL. Scherer, J. M., Stillwell, W., and Jenski, L. J. (1999). Anomalous changes in forward scatter of lymphocytes with loosely packed membranes. Cytometry 37(3), 184–190. Schlossman, S. F., Boumsell, L., Gilks, W., et al. (1994). CD antigens 1993. J. Immunol. 152(1), 1–2. Schmid, I., Krall, W. J., and Uittenbogaart, C. H. (1992). Dead cell discrimination with 7-aminoactinomycin D in combination with dual color immunofluorescence in single laser flow cytometry. Cytometry 13(2), 204–208. Schmitz, G., Wulf, G., Bruning, T., et al. (1987). Flow-cytometric determination of high-densitylipoprotein binding sites on human leukocytes. Clin. Chem. 33(12), 2195–2203. Schwartz, A., and Fernandez-Repollet, E. (1993). Development of clinical standards for flow cytometry. Ann. NY Acad. Sci. 677, 28–39. Schwartz, A., Marti, G. E., Poon, R., et al. (1998). Standardizing flow cytometry: A classification system of fluorescence standards used for flow cytometry. Cytometry 33, 106–114. Scott, J. E. (1972). Lies, dammed lies-and biological stains. Histochem. J. 4, 387–389. Serke, S., Lessen, A., and Huhn, D. (1998). Quantitative fluorescence flow cytometry: A comparison of the three techniques for direct and indirect immunofluorescence. Cytometry 33, 179–187. Severin, E., and Stellmach, J. (1984). Flow cytometry of redox activity of single cells using a newly synthesized fluorescent formazan. Acta Histochem. 75, 101. Sgonc, R., Boeck, G., Dietrich, H., et al. (1994). Simultaneous determination of cell surface antigens and apoptosis. TIG 10/2, 41–42. Sgonc, R., and Gruber, J. (1998). Apoptosis detection: An overview. Exp. Gerontol. 33(6), 525–533. Shankey, T. V., Rabinovitch, P. S., Bagwell, B., et al. (1993a). Guidelines for implementation of clinical DNA cytometry. International Society for Analytical Cytology. Cytometry 14, 472–477. Shankey, T. V., Kallioniemi, O. P., Koslowski, J. M., et al. (1993b). Consensus review of the clinical utility of DNA content cytometry in prostate cancer. Cytometry 14, 497–500. Shapiro, H. M. (1981). Flow cytometric estimation of DNA and RNA content in intact cells stained with Hoechst 33342 and pyronin Y. Cytometry 2, 143–145. Shapiro, H. M. (1994). Cell membrane potential analysis. Methods Cell Biol. 41, 121–133. Shapiro, H. M. (1995). “Practical Flow cytometry,” 3rd ed. Wiley-Liss, Inc., New York. Sharpless, T. K., Bartholdi, M., and Melamed, M. R. (1977). Size and refractive index dependence of simple forward angle scattering measurements in a flow system using sharply focused illumination. J. Histochem. Cytochem. 25, 845–856.

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

295

Sharpless, T. K., and Melamed, M. R. (1976). Estimation of cell size from pulse shape in flow cytofluorometry. J. Histochem. Cytochem. 24, 257–264. Sharpless, T. K., Traganos, F., Darzynkiewicz, Z., et al. (1975). Flow cytofluorimetry: Discrimination between single cells and cell aggregates by direct size measurements. Acta Cytol. 19, 577–581. Shinitzky, M., and Henkart, P. (1979). Fluidity of cell membranes-current concepts and trends. Int. Rev. Cytol. 60, 121–147. Shinitzky, M., and Inbar, M. (1976). Microviscosity parameters and protein mobility in biological membranes. Biochim. Biophys. Acta 433, 133–149. Silver, R. B. (1998). Ratio imaging: Practical considerations for measuring intracellular calcium and pH in living tissue. Methods Cell Biol. 56, 237–251. Simmons, D. M., Mercer, A. V., and Hallas, G. (1990). Characterization of six textile dyes as fluorescent stains for flow cytometry. J. Histochem. Cytochem. 38(1), 41–49. Sims, P. J., Waggoner, A. S., Wang, C. H., et al. (1974). Studies on the mechanism by which cyanide dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles. Biochemistry 13, 3315–3330. Smiley, S. T., Reers, M., Mottola-Hartshorn, C., et al. (1991). Intracellular heterogeneity in mitochondrial membrane potential revealed by a J-aggregate forming lipophilic cation JC-1. Proc. Natl. Acad. Sci. USA 88, 3671–3675. Stokke, T., and Steen, H. B. (1987). Distinction of leukocyte classes based on chromatin-structuredependent DNA-binding of 7-aminoactinomycin D. Cytometry 8, 576–583. Steinkamp, J. A., Fulwyler, M. J., Coulter, J. R., et al. (1973). A new multiparameter separator for microscopic particles and biological cells. Rev. Sci. Instrum. 44, 1301–1310. Steinkamp, J. A., and Crissman, H. A. (1993). Resolution of fluorescence signals from cells labeled with fluorochromes having different lifetimes by phase-sensitive flow cytometry. Cytometry 14, 210–216. Steinkamp, J. A., and Kraemer, P. M. (1974). Flow microfluorimetric studies of lectin binding to mammalian cells. II. Estimation of the surface density of receptor sites by multiparameter analysis. J. Cell Physiol. 84, 197–204. Steinkamp, J. A., Wilson, J. S., Saunders, G. C., et al. (1982). Phagocytosis: Flow cytometric quantitation with fluorescent microspheres. Science 215, 64–66. Stephenson, R. A., Gay, H., Fair, W. R., Melamed, M. R. (1986). Effect of section thickness on quality of flow cytometric DNA content determinations in paraffin-embedded tissues. Cytometry 7, 41–44. Stryer, L., and Haugland, R. P. (1967). Energy transfer: A spectroscopic ruler. Proc. Natl. Acad. Sci. USA 58, 719. Streicher, J., Valent, P., and Schmidt, H. (1999). Up-regulation of LDL-receptor expression by LDLimmunoapheresis in patients with familial hypercholesterolemia. J. Invest. Med. 47(8), 378–387. Suzuki, K., Ehara, T., Osafune, T., et al. (1994). Behavior of mitochondria, chloroplasts and their nuclei during the mitotic cycle in the ultramicroalga Cyanidioschyzon merolae. Eur. J. Cell Biol. 63(2), 280–288. Sweet, R. G. (1965). High frequency recording with electrostatically deflected ink jets. Rev. Sci. Instrum. 36, 13. Szollosi, J., Damjanovich, S., Balazs, M., et al. (1989). Physical association between MHC class I and class II molecules detected on the cell surface by flow cytometric energy transfer. J. Immunol. 143, 208–213. Szollosi, J., Damjanovich, S., and Matyus, L. (1998). Application of fluorescence resonance energy transfer in the clinical laboratory: Routine and research. Cytometry 34(4), 159–179. Szollosi, J., Matyus, L., Tron, L., et al. (1987). Flow cytometric measurements of fluorescent energy transfer using single laser excitation. Cytometry 8, 120–128. Szollosi, J., Tron, L., Damjanovich, S., et al. (1984). Fluorescence energy transfer measurements on cell surfaces: A critical comparison of steady-state fluorimetric and flow cytometric methods. Cytometry 5, 210–216.

10/31/2000

05:27 PM

296

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

Tanke, H. J., Niewenhuis, I. A. B., Koper, G. J. M. (1981). Flow cytometry of human reticulocytes based on RNA fluorescence. Cytometry 1, 313–320. Tanke, H. J., and van der Keur, M. (1993). Selection of defined cell types by flow-cytometric cell sorting. Trends Biotechnol. 11(2), 55–62. Taylor, I. W. (1980). A rapid single step staining technique for DNA analysis by flow microfluorimetry. J. Histochem. Cytochem. 28, 1021–1024. Teague, K., and El-Naggar, A. (1994). Comparative flow cytometric analysis of proliferating cell nuclear antigen (PCNA) antibodies in human solid neoplasms. Cytometry 15, 21–27. Thorell, B. (1980). Intracellular red-ox steady states as basis for cell characterization by flow cytofluoremetry. Blood Cells 6, 745–751. Thornthwaite, J. T., Sugarbaker, E. V., and Temple, W. J. (1980). Preparation of tissues for DNA flow cytometric analysis. Cytometry 1, 229–237. Thornthwaite, J. T., Thomas, R. A., Russo, J., et al. (1985). A review of DNA flow cytometric preparatory and analytical methods. In (J. Russo, Ed.), “Immunochemistry in Tumor Diagnosis,” pp. 380, Martinus Nijhoff, Boston. Tiirikainen, M. I. (1995). Evaluation of red blood cell lysing solutions for the detection of intracellular antigens by flow cytometry. Cytometry 20, 341–348. Timm, E. A. Jr., Podniesinski, E., Duckett, L., et al. (1995). Amplification and detection of a Y-chromosome DNA sequence by fluorescence in situ polymerase chain reaction and flow cytometry using cells in suspension. Cytometry 22(3), 250–255. Traganos, F., Darzynkiewicz, Z., Sharpless, T., et al. (1977). Simultaneous staining of ribonucleic and deoxiribonucleic acids in unfixed cells using acridine orange in a flow cytofluorometric system. J. Histochem. Cytochem. 25, 46–56. Traill, K. N., Boeck, G., Winter, U., et al. (1986). Simple method for comparing large numbers of flow cytometry histograms exemplified by analysis of the CD4 (T4) antigen and LDL receptor on human peripheral blood lymphocytes. J. Histochem. Cytochem. 34(9), 1217–1221. Traill, K. N., Juergens, G., Boeck, G., et al. (1987a). High density lipoprotein uptake by freshly isolated human peripheral blood T lymphocytes. Immunobiology 175, 447–454. Traill, K. N., Juergens, G., Boeck, G. et al. (1987b). Analysis of fluorescent low density lipoprotein uptake by lymphocytes. Paradoxical incease in the elderly. Mech. Ageing Dev. 40, 261–288. Trask, B. J., Mefford, H., van den Engh, G., et al. (1996). Quantification by flow cytometry of chromosome-17 deletions in Smith-Magenis syndrome patients. Human Genet. 98(6), 710–718. Trask, B. J., van den Engh, G., Christensen, M., et al. (1991). Characterization of somatic cell hybrids by bivariate flow karyotyping and fluorescence in situ hybridization. Somat. Cell Mol. Genet. 17(2), 117–136. Treumer, J., and Valet, G. (1986). Flow-cytometric determination of glutathione alterations in vital cells by o-phthaldialdehyde (OPT) staining. Exp. Cell Res. 163, 518–524. Tribukait, B., Granberg-Ohman, I., and Wijkstrom, H. (1986). Flow cytometric DNA and cytogenetic studies in human tumors: A comparison and discussion of the differences in modal values obtained by the two methods. Cytometry 7, 194–199. Trinkle, L. S., Wellhausen, S. R., and McLeish, K. R. (1987). A simultaneous flow cytometric measurement of neutrophil phagozytosis and oxidative burst in whole blood. Diag. Clin. Immunol. 5, 62–68. Tron, L., Szollosi, J., and Damjanovich, S. (1987). Proximity measurements of cell surface proteins by fluorescence energy transfer. Immunol. Lett. 16, 1–9. Tsien, R. Y. (1998). The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544. Uckun, F. M., Fauci, A. S., Chandan-Langlie, M., et al. (1989). Detection and characterization of human high molecular weight B cell growth factor receptors on leukemic B cells in chronic lymphocytic leukemia. J. Clin. Invest. 84, 1595–1608. Ulicny, J. (1992). Lorenz-Mie light scattering in cellular biology. Gen. Physiol. Biophys. 11, 133– 151.

10/31/2000

05:27 PM

Cytology

PS012-05.tex

PS012-05.xml

FLOW CYTOMETRY IN CELL AND MOLECULAR BIOLOGY

LaTeX2e(2000/08/16)

Textures 2.0

297

Valet, G., Raffael, A., Moroder, L., et al. (1981). Fast intracellular pH determination in single cells by flow cytometry. Naturwissenschaften 68, 265–266. Valet, G., Raffael, A., and Russmann, L. (1985). Determination of intracellular calcium in vital cells by flow cytometry. Naturwissenschaften 72, 600–602. van den Engh, G. J., Trask, B. J., and Gray, J. W. (1986). The binding kinetics and interaction of DNA fluorochromes used in the analysis of nuclei and chromosomes by flow cytometry. Histochemistry 84(406), 501–508. van den Engh, G. J., Trask, B. J., and Visser, J. W. M. (1985). Flow cytometer for identifying algae by chlorophyll fluorescence. US Patent 4,500,641. van de Winkel, M., Maes, E., and Pipeleers, D. (1982). Islet cell analysis and purification by light scatter and autofluorescence. Biochem. Biophys. Res. Commun. 107(2), 525. Van der Goot, F. G., Seigneur, A., Gaucher, J. C., et al. (1992). Flow cytometry and sorting of amphibian bladder endocytic vesicles containing ADH-sensitive water channels. J. Membr. Biol. 128(2), 133– 139. Van Dilla, M. A. (1985). “Flow Cytometry: Instrumentation and Data Analysis.” Academic Press, New York. Van Dilla, M. A., Fulwyler, M. J., and Boone, I. U. (1967). Volume distribution and separation of normal human leucocytes. Proc. Soc. Exp. Biol. Med. 125, 367–370. Van Dilla, M. A., Langlois, R. G., Pinkel, D., et al. (1983). Bacterial characterization by flow cytometry. Science 220, 620–622. Van Dilla, M. A., Trujillo, T. T., Mullaney, P. F., et al. (1969). Cell microfluorometry: A method for rapid fluorescence measurement. Science 163, 1213–1214. Van Engeland, M., Nieland, L. W., Ramaekers, F. C., et al. (1998). Annexin V-affinity assay: A review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 31, 1–9. van Ewijk, W., van Soest, P. L., Verkerk, A., et al. (1984). Loss of antibody binding to prefixed cells: Fixation parameters for immunocytochemistry. Histochem. J. 16, 179–193. Vesely, R., Barths, J., Vanlangendonck, F., et al. (1996). Initial results of central european immunophenotyping quality control program (CEQUAL). Cytometry (Communications in Clinical Cytometry) 26, 108–112. Vielh, P. (1991). “Flow Cytometry. Guides to Clinical Aspiration Biopsy.” Igaku-Shoin, New York. Vindelov, L. L., and Christensen, I. J. (1990). A review of techniques and results obtained in one laboratory by an integrated system of methods designed for routine clinical flow cytometric DNA analysis. Cytometry 11(7), 753–770. Vindelov, L. L., Christensen, I. J., Jensen, G., et al. (1983c). Limits of detection of nuclear DNA abnormalities by flow cytometric DNA analysis. Results obtained by a set of methods for sample storage, staining and internal standardization. Cytometry 3, 332–339. Vindelov, L. L., Christensen, I. J., Keiding, N., et al. (1983a). Long-term storage of samples for flow cytometric analysis. Cytometry 3, 317–322. Vindelov, L. L., Christensen, I. J., and Nissen, N. I. (1983b). A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry 3, 323–327. Vindelov, L. L., Christensen, I. J., and Nissen, N. I. (1983c). Standardization of high-resolution flow cytometric DNA analysis by the simultaneous use of chicken and trout red blood cells as internal reference standards. Cytometry 3, 328–331. Visser, J. W. M., Jongeling, A. A. M., and Tanke, H. J. (1979). Intracellular pH-determination by fluorescence measurements. J. Histochem. Cytochem. 27, 32–35. Watson, J. V. (1980). Enzyme kinetic studies in cell populations using fluorogenic substrates and flow cytometric techniques. Cytometry 1, 143–151. Watson, J. V. (1992). “Flow Cytometry Data Analysis—Basic Concepts and Statistics.” Cambridge University Press, Cambridge, UK. Weier, H. U., Polikoff, D., Fawcett, J. J., et al. (1994). Generation of five high-complexity painting probe libraries from flow-sorted mouse chromosomes. Genomics 21(3), 641–644.

10/31/2000

05:27 PM

298

Cytology

PS012-05.tex

PS012-05.xml

LaTeX2e(2000/08/16)

Textures 2.0

GUENTHER BOECK

Weil, G. J., and Chused, T. M. (1981). Eosinophil autofluorescence and its use in isolation and analysis of human eosinophils using flow microfluorometry. Blood 57, 1099–1104. Wheeless, L. L., Patten, S. F., and Berkhan, T. K., et al. (1984). Multidimensional slit-scan prescreening system: preliminary results of a single-blind clinical study. Cytometry 5(1) Wheeless, L. L., Badalament, R. A., de Vere White, R. W., et al. (1993). Consensus review of the clinical utility of DNA cytometry in bladder cancer. Report of the DNA Cytometry Consensus Conference. Cytometry 14, 478–481. Williams, J. M., Shapiro, H. M., Milford, E. L., et al. (1982). Multiparameter flow cytometric analysis of lymphocyte subpopulation activation in lectin-stimulated cultures. J. Immunol. 128, 2676–2681. Wilson, H. A., and Chused, T. M. (1985). Lymphocyte membrane potential and Ca2+ -sensitive potassium channels described by oxonol dye fluorescence measurements. J. Cell Physiol. 125, 72–81. Wilson, H. A., Seligmann, B. E., and Chused, T. M. (1985). Voltage-sensitive cyanine dye fluorescence signals in lymphocytes: Plasma membrane and mitochondrial components. J. Cell Physiol. 125, 61– 71. Wilson, R. B., and Murphy, R. F. (1989). Flow-cytometric analysis of endocytic compartments. Methods Cell Biol. 31, 293–317. Wolfbeis, O. S., and Urbano, E. (1982). Syntheses of fluorescent dyes, XIV. Standards for fluorescence measurements in the near neutral pH-range. J. Heterocyclic Chem. 19, 841. Yentsch, C. M. (1990). Environmental health: Flow cytometric methods assess our water world. Methods Cell Biol. 33, 575–612. Zarrin, F., Risfelt, J. A., and Dovichi, N. J. (1987). Light scatter detection within the sheath flow cuvette for size determination of multicomponent submicrometer particle suspensions. Anal. Chem. 59, 850–854. Zatloukal, K., Boeck, G., Rainer, I., et al. (1991). High molecular weight components are main constituents of mallory bodies isolated with a fluorescence activated cell sorter. Lab. Invest. 64, 200–206. Zelenin, A. V., Poletaev, A. I., Stepanova, N. G., et al. (1984). 7-Amino-actinomycin D as a specific fluorophore for DNA content analysis by laser flow cytometry. Cytometry 5, 348–354. Zhu, J., Musco, M. L., and Grace, M. J. (1999). Three-color flow cytometry analysis of tricistronic expression of eBFP, eGFP, and eYFP using EMCV-IRES linkages. Cytometry 37, 51–59. Zola, H., Flego, L., and Sheldon, A. (1992). Detection of cytokine receptors by high-sensitivity immunofluorescence/flow cytometry. Immunobiology 185, 350–365.