Quantitation of molecular and cellular probes in populations of single cells using fluorescence

Molecular and Cellular Probes (1987) 1, 121-136

REVIEW

Quantitation of molecular and cellular probes in populations of single cells using fluorescence

James V. Watson MRC Clinical Oncology Unit, The Medical School, Hills Road, Cambridge CB2 2QH, UK (Received 26 March 1987, Accepted 1 May 1987)

KEYWORDS: fluorescence, flow cytometry, molecular probes .

INTRODUCTION

The use of fluorescence techniques for quantitating molecular and cellular probes has vast potential for understanding both normal and abnormal physiological processes in all branches of biological science, including medicine and pathology . Immunocytochemical localization of specific molecules using monoclonal antibodies with both peroxidase and fluorescence probes in tissue sections is familiar to all . Quantitation of the number of molecules of a specific type in a sample of known weight or volume using radioimmune and ELISA assays is equally familiar . So also is the determination of enzyme reaction kinetic parameters in homogenized preparations using a number of colorimetric and fluorescence methods . Colorimetric techniques include high-pressure liquid chromatography where detection of a given molecule or molecule type is dependent on light-absorption properties . However, it is not generally appreciated that fluorescence techniques have been developed for quantitating the number of molecules per cell on an individual cell basis at very rapid rates . These techniques require the use of flow cytometry (FCM) which has been used in a bewildering variety of quantitative assays . These include measurements of DNA, DNA plus RNA simultaneously, intracellular pH and calcium, external membrane potential, mitochondrial function, enzyme reaction kinetics of both intracellular and membrane-bound enzymes, specific molecules tagged with monoclonal antibodies, fluorescence emission spectrum analysis and quantitation of specific proteins, including those encoded by oncogenes, measured simultaneously with DNA . Apart from the variety and speed with which quantitative data can be obtained using flow cytometry it also has the advantage of statistical precision and extreme sensitivity with a detection limit of the order of 150 molecules of free 0890-8508/87/010121 + 16 $03 .00/0

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fluorescein per cell .' However, the most powerful advantages, which are unique to the technology, derive from the fact that multiple correlated simultaneous measurements are made on an individual cell basis . This enables the investigator not only to identify cell subsets within a heterogeneous population but also to sort these from the population and determine the distribution of molecules in cells of those subsets . The objective of this article is to introduce the reader to the technique of flow cytometry and to some of the probes and methods being used to quantitate various molecules and biochemical processes at the cellular and subcellular level using fluorescence .

THE TECHNOLOGY A single-cell suspension is streamed coaxially in a flow chamber so that individual cells pass single file through the focus of a high-intensity excitation light source . This gives rise to both scattered light from all cells and fluorescence from suitably stained constituents although, obviously, only those cells containing the specifically labelled fluorescent molecules will exhibit fluorescence signals . In practice, lasers are frequently used for excitation as the light beam is of sufficiently high intensity to excite enough fluorescence from each cell in the few microseconds taken to traverse the focal point and they give a very stable light output over many hours of continuous operation . Each flash of scattered and/or fluorescent light is picked up by a light collection optical system and filtered into specific colour wavelength bands with dichroic mirrors and band pass filters . The total quantity of light in each colour band is then converted into an electrical signal by photodetectors each of which responds within its specific colour band . The detectors can be either photomultiplier tubes or solid-state devices and the electrical signals from the detectors are amplified then converted to digital form by analogue-to-digital converters (ADC) and stored transiently in computer memory and thence to disk for subsequent display and analysis . Typically, up to 5000 cells can be analysed per second and with cell sorting flow cytometers between 3000 and 4000 cells can be separated and collected per second with >90% purity . Cell sorters were developed from ink jet writing with electrostatically charged droplets .' When fluid is forced through a nozzle the resulting jet breaks up into droplets . If the nozzle is oscillated at high frequency, usually at about 40,000 Hz, the jet stream will break up into regular droplets which are formed at the oscillating frequency . Furthermore, the point at which the droplets break off from the jet will occur at a constant distance from the nozzle if the flow rate is constant . If single cells are contained in the jet and are passing a given point at a rate of 5000 per sec we expect, on average, one droplet in eight to contain a single cell . The cell stream within the fluid flow is intersected by the exciting light source ('analysis point') at a fixed distance from the point at which the droplets break off from the jet ('sort decision point') . As the flow rate is fixed, the time taken for a given cell to travel from the 'analysis point' to the 'sort decision point' is also fixed . Thus, if a cell is analysed and found to have the pre-determined fluorescence and light-scattering characteristics in which the investigator is interested, an electrostatic charge can be placed on the stream just as that cell is



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surrounded by the next droplet being formed at the droplet break-off point . A split second later the cell is contained within an isolated charged droplet in free fall . Further down stream the charged droplet containing its cell passes constantly charged deflection plates which steer the droplet into a suitable collection vessel . Many commercial machines are now equipped with four detectors and two lasers for two- or three-colour fluorescence work . However, some multi-laser research and development instruments have up to eight detectors (Watson, Horsnell and Smith, in prep .) and one such instrument has collected data on 32 light-scatter detectors

simultaneously .3 Data-processing and display procedures have been developed which handle up to eight-dimensional fully cross-correlated data in a single pass through the data set using microcomputers with only 28K addressable memory (Watson, Hornell and Smith, in prep .) . Following collection and any processing that may be required the data are recalled from disk and displayed in any one of a number of forms . The simplest is the monodimensional histogram where the frequencies of given signal magnitudes are plotted against the magnitudes . Twodimensional data are presented as either dot-plots or contour maps of one measurement on the y-axis plotted against the second correlated measurement on the x-axis together with the associated monodimensional histograms . Gates can be set in such a 2-D data space so that the investigator can display third or fourth correlated measurements associated with the cells contained within the gated regions . Some three-dimensional algorithms are also available which display the correlated measurements from each cell as 'clouds' within a three-dimensional data space where the density of each cloud is proportional to frequency . These somewhat more sophisticated data presentation methods require 3-D stereo perspective computer graphics techniques, but in most cases such data can be appreciated using sequential 2-D displays .

DYES, STAINING AND APPLICATIONS Fluorescent dyes and staining techniques are legion and a detailed account is not indicated in this brief review . The reader is referred to an excellent 600-page volume' for such accounts . As yet, there is no universally satisfactory or accepted classification of stains and staining techniques . However, the methods can be divided into three operational categories . Firstly, those in which the fluorochrome is coupled covalently to antibodies to probe specific molecules . Fluorescein and rhodamine, emitting green and orange/red fluorescence respectively, have been most commonly used in these types of applications . More recently, phycoerythrin (blue light excited red fluorescence)' and aminomethyl coumarin acetic acid (UV light excited blue florescence)' have further increased the range of studies that can be contemplated using antibody probes . Secondly, fluorescent dyes can accumulate unchanged within the cell either non-specifically or due to binding with a cellular component . Examples include fluorescein isothiocyanate (FITC) which binds to proteins and diphenyl hexatriene (DPH) which partitions in lipids . Thirdly, there are fluorescent ligands which interact with a cellular constituent, or the microenvironment, to release the fluorophore or modulate its emission . This last category includes non-covalently binding nucleic acid dyes, membrane potential, pH and calcium probes, and fluorogenic substrates for measuring enzyme activities .



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(1) Nucleic acid dyes Nucleic acid dyes have been used extensively in FCM and include DNA and nucleic acid specific ligands and polyanion dyes such as acridine orange for monovariate and bivariate cell cycle analysis in both control populations and those perturbed by anti-cancer agents .',' Probes such as ethidium bromide and propidium iodide are highly polar and intercalate with double-stranded nucleic acids which enhances the red fluorescence emission of the fluorochrome very considerably . The ligand polarity results in exclusion from cells with intact functioning membranes and cells must not only be rendered permeable but also treated with ribonuclease to obtain quantitative DNA data . Figure 1 shows a simple example of flow cytometric analysis procedures from a population of cells treated with colcemid and stained for DNA with ethidium bromide . The top display shows 90° light scatter on the ordinate vs forward scatter on the abscissa as a contour plot. The monodimensional histograms for each measurement are shown against the respective axes . Clearly, there are two subsets, one defined by low 90° light-scatter signals . Two elliptically gated regions, R1 and R2, have been set in this data space and we can now determine the light scatter and DNA content associated with these two subsets . This is shown in the lower six panels which show the forward scatter, 90° scatter and DNA associated with each region . The DNA histograms are shown in the two lowest panels where it can be seen that the region 1 cells exhibit a DNA histogram where cells are distributed throughout the cell cycle . The first peak represents cells in G1, the second peak represents cells with a doubled DNA content which have entered G2 + M and the trough between these peaks represent cells at various stages of DNA synthesis . The region 2 cells have a G2 + M DNA content and represent cells arrested in metaphase . The polarity of these DNA dyes and their exclusion from viable cells can be used in viability and cell killing assays' where they are used in conjunction with fluorescenated (green fluorescence) monoclonal-antibody probes . Dead cells take up the DNA stain and fluoresce red but viable cells exclude the dye and are red fluorescence-negative . Hence, it is possible to obtain quantitative monoclonal antibody data in both the live- and dead-cell fractions . DNA fluorochromes are also used for chromosome analysis with both single and dual laser systems not only for automated karyotyping but also for constructing cloned gene libraries from individually sorted chromosomes .'' The single laser system uses ethidium bromide to stain total DNA per chromosome to give a monodimensional flow karyotype in which the majority of the human chrornosomes have been identified" and Fig . 2 shows such an example . The dual laser system uses a double fluorochrome staining technique, 12 one UV excited (Hoechst 33258) and the second mid-blue excited (chromomycin-A3) . Hoechst 33258 binds to repetitive A-T base sequences and chromomycin-A3 binds preferentially to G-C rich regions . The chromosomes first pass through the UV beam which is absorbed by only the Hoechst/A-T complex . The absorbed energy is then transferred to the chromomycin-A3 which emits green fluorescence in proportion to A-T content . On passing through the second laser beam (mid-blue) the chromomycin-A3 is excited directly and a second flash of green fluorescence is given out, but this time it is proportional to G-C content . Hence, a bivariate profile of each chromosome is obtained which contains A-T vs G-C data . Karyotypes so



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Fig. 1 . The top display shows 90° light scatter (R-scatter) on the ordinate vs forward scatter (F-scatter) on the abscissa as a contour map and the monodimensional histograms are shown against the respective axes . Two elliptically gated regions, R1 and R2, have been set in this data space and the light scatter and DNA content associated with these two subsets are shown in the lower six panels . The DNA histograms in the lowest two panels show that the region 1 cells exhibit a DNA content characteristic of cells distributed throughout the cell cycle . The first peak represents cells in G1, the second peak represents cells with a doubled DNA content which have completed synthesis and the trough between these peaks represents cells at various stages of DNA synthesis . The region 2 cells have a G2 + M DNA content and represent cells arrested in metaphase .

generated have resolved all the human chromosomes apart from the 9-12 group and this technique is also being used to sort chromosomes and to construct gene libraries . The bis-benzimidazoles, including Hoechst 33342, are able to traverse the external cell membrane and be used as vital dyes . They bind in the minor groove of



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Relative fluorescence Fig . 2 . Flow karyotype from the MRC-5 46,XY diploid fibroblast cell line with the chromosome numbers adjacent to each peak . Redrawn from the data of Davies et al .'° and reproduced by permission of the editor of Nature.

DNA and have interesting properties as the emission spectrum when bound to DNA is dependent on the DNA : dye ratio and on the pH of the microenvironment . At low dye : DNA ratios the fluorescence emission is short wavelength (violet) biased but at higher ratios the emission is long wavelength (red) biased ." These differences can be measured by using two or more detectors responding in different regions of the emission spectrum ." This phenomenon has enabled different subsets to be identified in heterogeneous populations including two subsets in chicken thymus (Fig . 3) and cells resistant to cytotoxic agents which excrete dye molecules by the same mechanisms used to excrete the drug . Cells resistant to the cytotoxic agent exhibit a violet biased Hoechst 33342 emission spectrum as the dye-to-DNA ratio is low . Acridine orange has the unusual property of giving rise to green fluorescence from double-stranded nucleic acids and red fluorescence from single strands and selective denaturation of double-stranded RNA gives a simultaneous quantitation of both nucleic acids ." This dye has been used extensively in experimental systems and has also been used clinically for leukaemia diagnosis and in a cervical-cancer pre-screening programme in conjunction with slit scanning to give nuclear/ cytoplasmic ratios . In blind trials a false negative rate of 018% with about a 10% false positive rate has been achieved ." Nuclei can be extracted from archival human biopsies for DNA ploidy studies using a number of fluorochromes including DAPI and ethidium bromide ." Such studies can reveal aneuploid components giving some prognostic information . 18 These techniques have been extended to include the simultaneous assay of DNA and nuclear-associated oncogene-encoded proteins" using propidium iodide for



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Fig. 3. Hoechst 33342 stained chicken thymocyte fluorescence excited by UV light and analysed simultaneously on the violet (ordinate) and green (abscissa) channels . Two major subsets are apparent being partly distinguished by different intensities of green fluorescence and being completely resolved in the two-dimensional violet-green data space . The proportions in each cluster correspond to those of cortical and medullary thymocytes .

DNA and a fluorescenated monoclonal antibody for the protein which were analysed on the red and green fluorescence channels respectively . There was a difference in nuclear p62 c-m y c levels comparing normal and neoplastic cells from cervical epithelium at P<0.00001 .20 These methods have now been adapted for

freshly fixed cells harvested with a brush biopsy technique which may be clinically useful for automation in cervical-cancer pre-screening . 21 The simultaneous DNA-p62' -myc assay has also been used to study the total

quantity and turnover of this protein in tissue culture cells in different phases of the cell cycle . 22 The protein level increased with fresh medium stimulation and

remained constant in all phases of the cell cycle (Fig . 4) but the half-life was found to be between 20 and 40 min in each cell cycle phase which paralleled the equally rapid turnover of the mRNA . The simultaneous determination of total and newly replicated DNA can also be assayed with similar types of techniques using anti-BrdU antibodies23 or biotinolated nucleotides . 24 The former technique has been used to obtain growth kinetic data from human head and neck tumours and the latter to study DNA replication in an in vitro synthesis system . An example of the latter is shown in Fig . 5(c) where total DNA is scored on the abscissa (propidium iodide, red fluorescence) with biotinolated nucleotide incorporation (fluorescein, green fluorescence) on the ordinate . Figure 5(a), (b) respectively show photomicrographs of DNA and biotin-associated fluorescence from the same population as was analysed flow cytofluorimetrically in Fig . 5(c) . The fluorescence microscope data show qualitatively that there are different degrees of biotin incorporation per nucleus and possibly some intensity variation of DNA fluorescence . The flow system data [Fig . 5(c)] show that the two are both directly and quantitatively related .

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Fig. 4. Simultaneous assay for DNA and the nuclear-associated c-myc oncogene-encoded protein p62 1-'Y' in stimulated mouse 3T3 cells . The left panel shows DNA (ordinate, red fluorescence) plotted against p621-`"Y c- associated fluorescence . The protein was assayed using a synthetic peptide-induced mouse monoclonal antibody . Panel B shows that pre-incubation of the antibody with the peptide used as the immunogen abolished subsequent antibody binding . This reproduction represents part of a figure previously published in the EMBO Journalz 2 and is reproduced with permission from the editor.

(2) Probes for enzyme activity Enzyme activity in individual cells can be determined in flow cytometry by either deposition of insoluble light-absorbing reaction products or by using fluorogenic substrates . The former techniques necessarily kill cells and are more difficult to control in flow cytometry than those based on fluorescence, now the preferred methods which have been reviewed in some detail .", "' Dolbeare and Smith 27 analysed peptidase activity by linking a peptide via the amino group of methoxynaphthylamine (MNA) to give peptidyl-MNA which is hydrolysed by the enzyme . The free amino group can then react with added 5-nitrosalicalaldehyde to form an insoluble complex with red fluorescence properties . This reaction was used to distinguish between intestinal crypt and villus cells in gut mucosa on their leucine aminopeptidase content which is not present in the stem cells of the crypts . Similar techniques using trapping agents with the reaction products of a number of substrates have been reviewed by Dolbeare and Smith . 28 Some of the first flow cytometric kinetic measurements of both cytoplasmic 29 and plasma membrane 30 enzymes were made with fluorogenic substrates based on fluorescein and methyl umberlifferones to give K m and Vmax for reactions in populations of intact cells . These methods, using fluorescein diacetate (FDA), 4methyl umbelifferyl acetate (MUA) and 3-0-methyl fluorescein phosphate (MR), all

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Fig . 5. Panels (a) and (b) respectively show photomicrographs of DNA and biotin-associated fluorescence. Nuclei show different degrees of biotin incorporation in (b) . There is also some variation in DNA staining intensity in (a) . The flow cytometric data in panel (c) show that biotin fluorescence (ordinate) is both directly and quantitatively related to total DNA (abscissa) . This figure is reproduced by permission of the editor of the EMBO Journal ."

non-fluorescent substrates from which the fluorochrome is released by hydrolysis, have the major advantage that cell viability is maintained . Also, by linking the time stamp from the computer into the data base, it was possible to obtain the enzyme progress curve directly by plotting fluorescence vs the time at which the measurement was made from each cell in the population . Such techniques measuring the hydrolysis of two non-specific esterase substrates (FDA and MUA) simultaneously on different colour channels, have been used to distinguish between different cell types on the relative rates at which the two substrates are hydrolysed by different esterases in the different cells ." The esterase assay has recently been extended to determine reaction inhibition induced by carbamoylating cytotoxic agents . 32 This is an indirect method for assaying the transport of such agents across the external membrane and of cytotoxic efficacy having entered the cell ." This is shown in Fig . 6 where the top two panels show three-dimensional displays of size vs fluorescence vs time for the control population (left panel) and a population treated with the nitrosourea, BCNU (right panel) . Note the decrease in fluorescence accumulation in the BCNU treated cells . This is summarized in the lower panel which shows the medians of the fluorescence distributions at different time intervals plotted against time . Similar assays have also been developed for membrane-associated y-glutamyl transferase to examine the relative resistances to aflatoxin B, and susceptibilities to



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y-glutamyl p-phenylene diamine mustard of enzyme-positive and negative hepatocyte cell lines . An enzymatic assay has recently been developed for intracellular

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glutathione (GSH) using monochlorobimane, mCIB (Workman & Watson, in prep .) . Reaction of mCIB with GSH is catalysed by the glutathione-S-transferases to generate a polar fluorescent conjugate which remains trapped within each cell due to the polarity of the molecule . The rate of reaction is a function of added mCIB, intracellular GSH and enzyme activity . Total cellular GSH is proportional to the asymptotic fluorescence level and enzyme activity is proportional to initial reaction rate . The technique is being used to investigate GSH metabolism kinetics in discrete



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subpopulations of heterogeneous tumour samples and the development of cellular resistance to anti-neoplastic drugs in those subsets .

(3) Membrane potential, pH and calcium probes Fluorescent membrane potential probes are charged symmetrical, 'mirror-image' ring-structure molecules with hydrocarbon side chains . The molecule retains lipophilicity as the charge is not localized and the relative lipophilicity can be engineered by changing the length of the side chains . The cationic cyanin dyes were the first such molecules to be recognized as membrane-potential probes . 35 They partition in lipids to an extent that varies partly with side chain length and partly with potential across the cell membrane . A second class of dyes, the oxanols, are functionally similar to the cyanins differing only in that they are anionic . 36 Cyanins are excluded as cells undergo depolarization whereas oxanols are excluded on reand hyper-polarization . The cyanins, however, suffer from the disadvantage that they are taken up extensively by mitochondria which can reduce the 'signal-tonoise' ratio when assessing external membrane potential . This problem, however, is overcome by using the oxanols . These probes have been used in studies of granulocyte and lymphocyte function and in mitogenic stimulation of T-cells . 37 Rhodamine-123 is another class of cationic dye which partitions into electro38,39 negative environments and has been described as a mitochondrial specific dye . This description was based on fluorescence microscopy observations which revealed intense mitochondrial staining and is not strictly correct as rhodamine-123 will partition into any electronegative compartment . However, the interior of mitochondria is very electronegative if the proton pump is functioning normally and staining intensity gives some indication of the integrity of normal mitochondrial function . Rhodamine-123 has been studied in some in vitro tumour cell lines where 4o it is reported to be preferentially retained and selectively cytotoxic . Positive ions can bind to a number of fluorochromes with a resulting modification of their electronic configuration which can lead to changes in either the absorption or emission spectrum . This phenomenon can be exploited in flow cytometry to give a measure of intracellular pH due to binding of H + ions . The absorption spectrum peak of one such probe, 6-carboxy fluorescein (6-CF), is close to the 458 nm argon line at low pH but increases to close to the 488 nm line at higher pH ;4' however, the position of the peak of the fluorescence emission shows little change with pH .42 Hence, by using two argon lasers tuned to these lines at the same power output and analysing the same cell sequentially we can obtain a ratio of the fluorescence emission at the two excitation wavelengths, and the ratio is proportional to pH . Two major problems arise with this approach . Firstly, 6-CF is highly polar and does not readily traverse the external cell membrane and secondly, the expense of two lasers tunable to the required wavelengths is considerable . The first problem is resolved by loading cells with the diacetate derivative, 6-CFDA, which is highly lipophilic and readily crosses the cell membrane . Intracellular esterases then release the 6-CF which remains trapped in the cell due to its polarity . Secondly, although the position at the peak of the emission spectrum of 6-CF does not change with pH, the relative magnitudes of the emission at 520 nm and 620 nm do show changes ."



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Thus, a measure of pH is obtained using a single laser excitation line by taking the ratio of the emission at these two different wavelengths . Valet et al ." developed a similar technique using the diacetyl derivative of 2,3-dicyano-1,4-dihydroxy benzene (DCDHB) which shows an emission spectrum shift from about 450 nm to 480 nm as pH increases .45 Examples of this type of assay are shown in Fig . 7 . Calcium participates in the regulation of many cellular functions including signal transduction across the external membrane,46 and a number of fluorescent probes which chelate calcium with high affinity have been developed . As with pH probes, the addition of positive Ca 2+ ions causes shifts in either the absorption or emission spectra which can be detected using similar techniques to those described above for pH measurement. These probes are all highly polar and are loaded into cells using the acetoxy derivatives which again rely upon esterases to release the probe . Quin-11 47 and its analogue FURA-11 48 exhibit absorption shifts on binding calcium . As with pH measurements this is not ideal for FCM ; however, INDO-1 48 shows an emission spectrum shift on binding Ca 2+ which can be monitored by simultaneous analysis at 400 nm and 480 nm . The ratio of the emission intensities at these wavelengths is proportional to the quantity of Ca z+ bound . This has been used to distinguish between T and B lymphocytes from mouse spleen cultures where the mixed population was subjected to B-cell mitogenic stimuli . Within 20 s the B-cell component had responded as indicated by their increase in Ca 2+ 4s

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(4) Monoclonal antibodies Fluorophore coupled monoclonal antibodies" have been used in conjunction with FCM in a simply enormous number of studies in both cell biology and medicine . Some have been mentioned in previous sections and even a brief review of this topic would fill the first year's issue of this journal . Perhaps the most widely studied are the haemopoetic and related systems . Automated typing of normal leucocytes, s ' and in leukaemias, immunosuppressed transplant patients and lymphomas are now routine services in many departments . The rapid progress in dissecting details of bone marrow lineages s2-sa during the 1980s has been due to development of suitable monoclonal antibodies and cell sorting flow cytometry .

CONCLUSIONS I hope it is self-evident from this brief review that the potential for fluorescence quantitation of cellular and molecular probes with flow cytometry is almost limitless . Indeed, with developments currently taking place the detection limit will be decreased to a few tens of molecules per cell, and when this is reached we will be limited only by our imaginations . Above all, however, this technology allows us to measure physiological and pathological processes in populations of single cells and in subsets of those populations very rapidly . This in turn enables us to test ideas and hypotheses that we could not previously contemplate testing and rapidly to find flaws in those ideas and hypotheses .

ACKNOWLEDGEMENTS I thank my colleagues and collaborators, J . Julian Blow, Stephen H . Chambers, Caroline Dive, Gerard I . Evan, David Hedley, Elizabeth Musgrove, Alexander Nakeff, Pamela Rabbitts, Terence H . Rabbitts, Paul J . Smith, Brian D . Young and Paul Workman and the editors of Biochemical Pharmacology, Cytometry, the EMBO Journal and Nature for permission to reproduce various of the figures used as illustrations .

REFERENCES 1 . Watson, J.V . & Walport, M.J . (1986) . Molecular calibration in flow cytometry with sub-attogram detection limit . Journal of Immunological Methods 93, 171-5 . 2 . Sweet, R .G . (1965) . High frequency recording with electrostatically deflected ink jets . Review of Scientific Instruments 36, 131 . 3 . Salzman, G .G., Crowell, J .M ., Goad, C .A ., Mullaney, P.F. & Coulter, J .R . (1975) . A flow-system multiangle light scattering instrument for cell characterization . Clinical Chemistry 21, 1297-304. 4. Lansing-Taylor, D ., Waggoner, A.S ., Murphy, R .F ., Lanni, F. & Birge, R .R . (eds) (1986) . Applications of Fluorescence in the Biomedical Sciences . New York: Alan R . Liss . 5 . Glazer, A .N . & Stryer, L . (1984) . Phycofluor probes . Trends in Biolochemical Sciences 9, 423-7. 6 . Khalfan, H ., Abuknesha, R., Rand-Weaver, M ., Price, R .G . & Robinson, D . (1986) . Aminomethyl coumarin acetic acid : a new fluorescent labelling agent for proteins . Histochemical journal 18, 497-9 . 7. Smith, P.J ., Anderson, C .O . & Watson, J .V . (1985) . Abnormal retention of X-irradiated ataxiatelangectasia fibroblasts in G2 phase of the cell cycle : cellular RNA content, chromatin stability and effects of 3-aminobenzamide. International Journal of Radiation Biology 47, 701-12 .



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8 . Smith, P.J ., Anderson, C .O . & Watson, J .V . (1985) . Effects of X-irradiation and sodium butyrate on cell-cycle traverse of normal and radiosensitive lymphoblastoid cells . Experimental Cell Research 160, 331 . 9 . Muirhead, K ., Horan, P .K . & Proste, G . (1985) . Flow cytometry : present and future . Biotechnology 3, 337-56 . 10 . Davies, K .E ., Young, B .D ., Elles, R .G ., Hill, M .E. & Williamson, R . (1981) . Cloning of a representative genomic library of the human X chromosome after sorting by flow cytometry . Nature (London) 293, 374-6 . 11 . Sillar, R . & Young, B .D . (1981) . A new method for the preparation of metaphase chromosomes for flow analysis. journal of Histochemistry and Cytochemistry 29, 74-8 . 12 . Langlois, R .G ., Yu, L .C., Gray, J .W . & Carrano, A .V . (1982) . Quantitative karyotyping of human chromosomes by dual laser beam flow cytometry . Proceedings of the National Academy of Science USA 79, 7867-80 . 13 . Smith, P . J . Nakeff, A . & Watson, J .V . (1985) . Flow-cytometric detection of changes in fluorescence emission spectrum of a vital DNA-specific dye in human tumour cells . Experimental Cell Research 159, 37-46 . 14 . Watson, J .V ., Nakeff, A., Chambers, S .H. & Smith, P.J . (1985) . Fluorescence emission spectrum analysis of chicken thymocytes using Hoechst-33342 . Cytometry 6, 310-15 . 15 . Traganos, F ., Darzynkiewicz. Z ., Sharpless, T . & Melamed, M .R . (1977) . Simultaneous staining of ribonucleic and deoxyribonucleic acid in unfixed cells using acridine orange in a flow cytofluorimetric system . Journal of Histochemistry and Cytochemistry 25, 46-56. 16 . Wheeless, L .L ., Patten, S .F ., Berkan, T .K ., Brookes, C .L., Gorman, K.M ., Lesh, S .R., Lopez, P .A . & Wood, J.C. (1984). Multidimensional slit-scan prescreening system : preliminary results of a single blind clinical study . Cytometry 5, 1-8 . 17 . Hedley, D .W ., Friedlander, M .I ., Taylor, LW., Rugg, C .A. & Musgrove, E.A . (1983) . Method for analysis of cellular DNA content of paraffin-embedded pathological material using flow cytometry . Journal of Histochemistry and Cytochemistry 31, 1333-5 . 18 . Friedlander, M .L ., Hedley, D .W . & Taylor, I .W . (1984). Clinical and biological significance of aneuploidy in human tumours-a review . journal of Clinical Pathology 37, 961-74 . 19 . Watson, J .V ., Sikora, E .K . & Evan, C .I . (1985) . A simultaneous flow cytometric assay for c-myc oncoprotein and cellular DNA in nuclei from paraffin embedded material . Journal of Immunological Methods 83, 179-92 . 20 . Hendy-lbbs, P ., Cox, H ., Evan, G .I . & Watson, J .V. (1987) . Flow cytometric quantitation of DNA and c-myc oncoprotein in archival biopsies of uterine cervix neoplasia . British journal of Cancer 55, 275-82 . 21 . Elias-Jones, J., Hendy-Ibbs, P., Cox, H ., Evan, G .I . & Watson, J .V . (1986). Cervical brush biopsy specimens suitable for DNA and oncoprotein analysis using flow cytometry . Journal of Clinical Pathology 39, 577-81 . 22 . Rabbitts, P .H ., Watson, J .V ., Lamond, A ., Fischer, W., Forester, A ., Stinton, M .A ., Evan, G .I ., Atherton, E ., Sheppard, R .C . & Rabbitts, T .H . (1985) . Metabolism of c-myc gene products: c-myc mRNA and protein expression in the cell cycle . EMBO Journal 4, 2009-15 . 23 . Gratzner, H .G . (1982) . Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine : a new reagent for detecting DNA replication . Science 218, 474-5 . 24. Blow, J .J . & Watson, J .V . (1987) . Nuclei act as independent and integrated replication units in a xenopus cell-free DNA replication system . EMBO Journal (in press) . 25 . Dolbeare, F .A . (1981) . Fluorogenic quantification of specific chemical species in single cells . In Modern Fluorescence Spectroscopy . (Wehry, E .L., ed .) pp . 251-93 . New York : Plenum Press . 26. Watson, J .V . (1984) . Flow cytometric methods for studying enzyme activity in populations of individual cells . In Antitumour Drug Resistance . (Fox, B.W. & Fox, M ., eds) pp . 187-203 . Berlin, Heidelberg, New York, Tokyo : Springer-Verlag . 27 . Dolbeare, F.A . & Smith, R .E . (1977) . Flow cytometric measurement of peptidases with the use of 5nitrosalicylaldehyde and 4-methoxy-ß-naphthylamine derivatives . Journal of Clinical Chemistry 23,1485-91 . 28 . Dolbeare, F .A . & Smith, R .E . (1979) . Flow cytoenzymology: rapid enzyme analysis of single cells . In Flow Cytometry and Sorting . (Melamed, M .R., Mullaney, P .F. & Mendelsohn, M.L ., eds) pp . 317--33 . New York : John Wiley .



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29 . Watson, J .V., Chambers, S .H ., Workman, P . & Horsnell, T.S. (1977). A flow cytometric method for measuring enzyme reaction kinetics in populations of single cells . FEBS Letters 81, 397 . 30 . Watson, J .V ., Workman, P . & Chambers, S .H. (1979) . An assay for plasma membrane phosphatase activity in populations of individual cells . Biochemical Pharmacology 28, 821-9. 31 . Watson, J .V . (1980). Enzyme kinetic studies in cell populations using fluorogenic substrates and flow cytometric techniques . Cytometry 1, 143 . 32 . Dive, C ., Workman, P . & Watson, J .V . (1987) . Improved methodology for intracellular enzyme reaction and inhibition kinetics by flow cytometry . Cytometry (in press). 33 . Dive, C ., Workman, P. & Watson, J .V . (1987) . Novel dynamic flow cytoenzymological determination of intracellular esterase inhibition by BCNU and related isocyanates. Biochemical Pharmacology (in press) . 34 . Manson, M.M., Legg, R .F ., Watson, J .V ., Green, J .A . & Neale, G.E. (1981) . An examination of the relative resistances to aflatoxin B, and susceptibilities to y-glutamyl p-phenylene diamine mustard of y-glutamyl transferase negative and positive cell lines . Carcinogenesis 2, 661-70 . 35. Sims, P.J ., Waggoner, A .S., Wang, C .H . & Hoffman, J .S . (1974) . Studies on the mechanism by which cyanin dyes measure membrane potential in red blood cells and phosphatidyl cholinic vesicles . Biochemistry 13, 3315-30 . 36. Rink, T .J ., Montecucco, C ., Hesketh, T .R . & Tsien, R.Y . (1980) . Lymphocyte membrane potential assessed with fluorescent probes . Biochimica et Biophysica Acta 595, 15-30. 37 . Tsien, R.Y ., Pozzan, T. & Rink, T .J . (1984) . T-cell mitogens cause early changes in cytoplasmic free Ca" and membrane potential in lymphocytes . Nature (London) 295, 68-70 . 38. Johnson, L .V ., Walsh, M .L ., Bockus, B .J . & Chen, L .B . (1981). Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy . Journal of Cell Biology 88, 526-35 . 39. Weiss, M .J. & Chen, L .B . (1984). Rhodamine 123 : a lipophilic mitochondrial-specific vital dye . Kodak Laboratory Chemical Bulletin 55, 1-4. 40. Lampidis, T .J ., Bernal, Summerhays, I .C . & Chen, L .B . (1982) . Rhodamine 123 is selectively toxic and preferentially retained in carcinoma cells in vitro . Annals of the New York Academy of Science 397,299-302 . 41 . Thomas, J .A., Buschbaum, R .N ., Zimmick, A . & Racker, E. (1979) . Intracellular pH measurements in Ehrlich ascites tumour cells utilizing spectroscopic probes generated in situ . Biochemistry 18, 2210-18 . 42 . Martin, M.M. & Linquist, L. (1975) . The pH dependence of fluorescein fluorescence . Journal of Luminescence 10, 381-90. 43 . Musgrove, E ., Rugg, C . & Hedley, D . (1986). Flow cytometric measurement of cytoplasmic pH : a critical evaluation of probes . Cytometry 7, 347-55 . 44 . Valet, G ., Raffael, A ., Moroden, L ., Wursch, E . & Ruhenstroth-Bauer, G . (1981) . Fast intracellular pH determination in single cells by flow cytometry . Naturweiss 68, 265-6 . 45 . Kurtz, I . & Balaban, R .S . (1985) . Fluorescence emission spectroscopy of 1,4-dihydroxyphtholonitrile . Biophysical journal 48, 499-508 . 46 . Campbell, A .K . (1983). Intracellular Calcium. New York : John Wiley and Sons . 47 . Tsien, R .Y . (1980) . New calcium indicators and buffers with high selectivity against magnesium and protons : design, synthesis and properties of prototype structures . Biochemistry 19, 2396-404 . 48 . Grynkiewicz, G ., Poenie, M . & Tsien, R.Y . (1985). A new generation of Ca" indicators with greatly improved fluorescence properties . Journal of Biological Chemistry 260, 3440-50 . 49 . Chused, T .M ., Wilson, A .H ., Seligman, B .E . & Tsien, R .Y . (1986) . Probes for use in the study of leukocyte physiology by flow cytometry . In Applications of Fluorescence in the Biomedical Sciences . (Lansing-Taylor, D ., Waggoner, A .S ., Murphy, R .F ., Lanni, F . & Birge, R .R ., eds) pp . 531-44 . New York: Alan R . Liss . 50. Kohler, G . & Milstein, C . (1975) . Continuous cultures of fused cells secreting antibody of predefined specificity . Nature (London) 256, 495-6 . 51 . Loken, M .R . (1986) . Cell surface antigen and morphological characterization of leukocyte populations by flow cytometry . In Monoclonal Antibodies . (Beverly, P.C.L ., ed .) . Methods in Haematology 13, 132-44 . 52 . Nicola, N .A., Metcalf, D., von Melcher, H . & Burgess, A .W . (1981) . Isolation of murine foetal hemopoetic progenitor cells and selective fractionation of various erythroid precursors . Blood 58, 376-86 .



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53 . Hoang, T ., Gilmore, D ., Metcalf, D ., Cobbold, S., Watt, S .M ., Clark, M .R . & Waldmann, H . (1983) . Separation of hemopoetic cells from adult mouse marrow by use of monoclonal antibodies . Blood 61,580-8 . 54 . Watt, S .M ., Gilmore, D .J ., Clark, M .R ., Davis, J .M., Swirsky, D .M . & Waldmann, H . (1984) . Haemopoetic progenitor cell heterogeneity revealed by a single monoclonal antibody, YW 13.1 .1 . Molecular Biology and Medicine 2, 351-68 .