Usage of the flow cytometer-cell sorter

Journal o f Irnmunological Methods, 47 (1981) 13--24 Elsevier/North-Holland Biomedical Press

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Review article USAGE O F THE FLOW CYTOMETER-CELL S O R T E R

R.G, MILLER, M.E. LALANDE, M.J. McCUTCHEON, S.S. STEWART and G.B. PRICE The Ontario Cancer Institute, 500 Sherbourne St., Toronto, Ont. M 4 X 1K9, Canada (Received 27 May 1981, accepted 10 August 1981}

Flow cytometer-cell sorters are playing an increasingly important role in research in cellular immunology. We here review operating procedures and applications developed for our home-built flow cytometer-cell sorter which may be of general interest.

INTRODUCTION

In many problems in cell biology and immunology, one wishes to identify and separate various functionally homogeneous subpopulations of cells conrained in a heterogeneous cell suspension. One approach has been to identify and separate cells on the basis of differences in their physical properties such as size, density and surface charge (Shortman, 1972; Miller et al., 1975; Miller, 1977). Although this approach has had some striking successes, it is useful only to the extent that the functional property of interest is associated with the physical separation variable used. What is really needed is methods of cell separation which recognize variables more closely related to cell function. The flow cytometer-cell sorter (FC-CS) appears to provide the underlying technology for a n u m b e r of such methods in that it enables the identification and separation o f individual cells on the basis o f biologically specific markers. • In all widely used cell separation procedures, except cell sorting, cells are processed as one bulk sample. By contrast, in cell sorters, cells are examined one at a time and a decision made for each cell as to whether or n o t it is to be sorted. This is simultaneously the greatest strength and the greatest weakness of the cell sorter. On the strength side, several different parameters, such as cell size, presence of 1 or 2 different surface markers, a m o u n t o f DNA, etc. can be measured for each cell and the sorting decision be made on the basis o f any combination o f these parameters. Bulk separation procedures are incapable of this kind of resolution. On the weakness side, examining cells one at a time is slow. Typically, rates are not much more than 103 cells/sec and are certainly less than 104 cells/sec if one wishes to maintain m a x i m u m purity. Taking this upper limit, it would require 10 s sec or 1.2 days to process 109 cells! In contrast, a bulk separation procedure, such as velocity sedimentation (Miller and Phillips, 1969), can easily handle 0022-1759/81/0000---0000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press

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Fig. 1. Detector configurations used for fluorescence (a) or polarization (b) analysis in our FC-CS as viewed from above with the laser beam coming in from the left. A, B, 2 cylindrical lenses used to focus the beam into an ellipse with the narrow axis oriented vertically; C, vertical jet of fluid containing cells confined to axis region; D, black lightabsorbing strips mounted in front of detectors so as to absorb either the direct laser beam (scatter detector) or laser light diffracted from the fluid jet (all detectors); E, objective lens for the scatter detector; F, variable thickness of translucent plastic sheet added to reduce intensity of signal to scatter detector as required; G, scatter detector (United Detector Technology Inc. PIN-10 Schottky barrier photocathode); H, I, lenses for fluores-

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109 cells in 2--4 h. Clearly, the analysis procedures used for sorted cells must be based on assays using relatively small numbers o f cells. Biochemical procedures requiring an input of 1 g of cells or more cannot realistically be approached. In vivo cell transfer experiments are often also precluded b u t in vitro assessment o f cell function is often possible. Sorting experiments can often be made more feasible if a bulk separation procedure is first used to enrich for the cell of interest or if cells are run twice through the sorter, the first time to obtain enrichment and the second to obtain purity. Since the development of the first practical FC-CS (Bonner et al., 1972), sorters have come into relatively widespread use and are n o w available commercially from several companies (Becton-Dickinson, Coulter Electronics, Ortho Pharmaceutical). Several reviews describe the technological aspects of cell sorting (Horan and Wheeless, 1977; Arndt-Jovin and Jovin, 1978; Miller and Price, 1979) and these will n o t be gone into here. Loken and Herzenberg (1975) have described several procedures for the operation and calibration of an FC-CS which we have found useful. Thus we describe only some special operating procedures we have developed. Although our system (Price et al., 1977; McCutcheon, 1979; Stewart et al., 1980) is home-built and therefore unique, these procedures should be readily adaptable to other systems. We then describe our m e t h o d o l o g y in 3 different application areas: surface labeling, cytoplasmic fluorescence depolarization and the use of Hoechst 33342 dye to identify cell subpopulations differing in membrane transport properties. All these procedures yield viable cells whose function can be assessed in in vitro culture assays. Non-vital staining procedures, e.g., use of mithramycin to measure DNA content (Crissman et al., 1979), are not described here. OPERATING PROCEDURES

General

Fig, l a shows the detector configuration for our FC-CS when used for analysis and sorting on the basis of fluorescence and light scattering intensity. The configuration is similar in principle to that of commercial instrumentation. The configuration used for the measurement of fluorescence

cence detector telescope; J, K, filters for exclusion of scattered laser light. For detecting fluorescence from fluorescein exited at 488 nm, we use a 530 nm (J) cut-on dyed glass filter (OG530, Jenaer Glaswerk Schott and Gen., Mainz, F.R.G.) in combination with a 560 nm (K) cut-off filter (Ditric Optics Inc., Marlboro, MA) to reduce filter fluorescence effects on signals from surface-labeled cells; L (Fig. l b only), polaroid filters oriented to permit passage of the fluorescent light polarized parallel to the incident laser beam in one detector and perpendicular to it in the other; M, photocathode of photomultiplier tube (RCA 4526); N, light-tight housing; O, filter to block scattered laser light; P, telescope to enable operator to observe laser-jet interaction, droplet formation, etc. Design details for the FC-CS system are given elsewhere (Price et al., 1977; McCutcheon, 1979; Stewart et al., 1979, 1980).

16 polarization is shown in Fig. l b , the principal differences being the addition of a second photomultiplier and the replacement of each 560 nm cut-off filter (Fig. l a , K) with an appropriately oriented polaroid filter (Fig. l b , L). In b o t h configurations, signals from all detectors are pre-amplified and passed through discriminators; only signals above some adjustable threshold level are recognized and subject to further processing. Such signals are applied to a single-channel analyzer (SCA) which generates an o u t p u t only if the magnitude of the applied signal lies within a 'window' of interest selected by the operator. All signals brighter than the threshold are available as analogue pulses, which can be accumulated in a histogram of cell number versus brightness by a multichannel analyzer (MCA). The MCA can be gated by the SCA signals to produce histograms of, say, fluorescence intensity from all cells which have light-scatter intensity within a given range of interest. When fluorescence polarization is being measured, signals (Ii,I2) from the two fluorescence detectors are applied to a c o m p a c t analogue computer which calculates a polarization value for each cell in real-time (Stewart et al., 1980). The above instrumentation is sufficient for analytical use of the FCCS (e.g., to measure the fraction of a cell population carrying a given fluorescent label or to measure the spectrum of polarization values in a population). For sorting applications, there are some additional instrumentation and operation requirements. When a cell exceeds the light-scatter threshold level, it is considered for sorting, and all other events are 'locked out' while the circuitry is busy processing this event. Signals n o t exceeding this threshold level (usually set to exclude cell debris) are ignored. Logic signals from all SCAs are tested for coincidence, and used to decide into which pool a cell is to be sorted, or whether to suppress the sorting event when cells are so close together that sorting either one would result in carrying the other along.

Maintenance of sterility When the system is not in use, the cell sorter fluid pathways are kept sterile b y filling them through the sheath flow system with a 0.10% v/v solution of commercial laundry bleach (5.25% sodium hypochlorite solution) in distilled water. Before a run, all lines are flushe~! free of bleach solution with sterile phosphate-buffered saline (PB8). During a run, the same sterile PBS is used to provide the sheath flow and is introduced through a disposable 0.22 ~m pore size Millipore filter. This appears sufficient to prevent bacterial or fungal infection from developing in the fluid pathways. Cells are collected in 1.5 ml polypropylene centrifuge tubes containing a preload o f 0.2 ml tissue culture medium with 10% FCS added. After sorting, cells may be grown in our standard culture medium supplemented with 50 ~g/ml gentamycin to suppress any residual bacterial contamination.

Sample preparation and loading 'Experience has shown that failure to obtain reliable and reproducible

17 data has more often resulted from poorly prepared samples then faulty or defective instrumentation' (Crissman et al., 1975). The most important requirements of a cell sample for flow c y t o m e t r y are that it be monodisperse (few doublets or larger multiplets) and free of damaged cells, cell debris, and large excesses o f uninteresting cells (e.g. red cells in a l y m p h o c y t e preparation). It is good practice to keep a microscope close to the FC-CS and to verify sample state routinely before sorting or analysis. It is amazing how often a sample said to contain 5 × 106 monodisperse cells/ml, 90% viable, is presented to the FC-CS with 103 cells/ml, 30% viable with most of the cells in aggregates. While it is not too difficult to prepare a clean suspension of cells labeled with a single or multiple step fluorescent a n t i b o d y stain, the requirements are n o t necessarily the same as for fluorescence microscopy. In particular, it is imperative that the suspension medium be as free as possible of u n b o u n d fluorescent material. Extraneous fluorescence from this source greatly increases the low-level noise. This is very serious when using rather dim label typical of most surface labels. Layering the cell suspension over and centrifuging the cells down through a small quantity of serum or dense (p = 1.04) BSA solution (Shortman et al., 1972) before the final wash is quite effective in reducing the problem. Our flow cytometer, apparently in c o m m o n with most others, at first used a differential pressure arrangement between core and sheath reservoirs to drive the cell suspension through the core flow line. This has two disadvantages. First, the core-sheath pressure balance is quite critical. If a glass capillary-array filter (Mosaic Fabrications, Bendix Corp., Sturbridge, MA)is used in the core line to prevent cell clumps from reaching the aperture, even a small pressure drop at the filter produced by partial blocking with cells a n d / o r debris can cause a large drop in the core flow-rate. Secondly, good optical resolution requires that the core flow be kept below a critical diameter in the sensing zone (McCutcheon and Miller, 1979). This diameter in turn depends on the core volume rate of flow. This is n o t easily directly determined in a differential pressure system and one tends to adjust it on the basis o f cell c o u n t rate. With varying cell concentrations in different samples, this can lead to large and undesirable variations in the core volume rate of flow. We have found that a constant displacement core flow system is simpler to use and produces more constant flow conditions than a differential pressure system. A commercial stepping-motor driven syringe pump (Model 352, Sage Instruments, White Plains, NY) is used to inject the cell sample at a selectable flow rate (ml/h). This rate remains constant with even major variations in back pressure due to, for example, the presence o f a capillaryarray filter in the core line. It is easily determined what upper limit on core flow rate can be allowed before resolution is degraded and sample concentration is adjusted to allow a useful cell flow rate at or below this critical volume rate of flow. An additional advantage is t h a t cell samples m a y be loaded in sterile disposable syringes. It is n o t difficult to adapt the pump to a refrigerated collar or housing for the cell sample if necessary.

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Optical resolution We have studied the factors involved in good fluorescence intensity resolution in stream-in-air flow sorters (McCutcheon and Miller, 1979). One critical area, and perhaps the most easily modified in an existing device, is the optical filter train. Resolution of bright fluorescence signals is limited by the c y t o m e t e r optical and hydraulic design, but resolution of dimmer fluorescent signals (typical of m a n y surface labels) is limited by photoelectron statistics. Hence it is important to choose filters which transmit as much as possible of the desired fluorescent light (typically 15--50% for fluorescein fluorescence excited by the 488 nm line from an argon laser) while minimizing laser light transmission. Dyed glass filters are prone to fluoresce themselves under laser irradiation. If such filters are employed, it may be necessary to use subsequent long-wavelength c u t o f f filters to remove as much as possible of the filter fluorescence, which can contribute a good deal of noise in the measurement of weak signals, and ultimately limit the ability to detect cells with small amounts of label. The filter combination we routinely use for detecting the fluorescence of cell-surface-labeled fluorescein excited with the 488 nm laser line is indicated in Fig. la.

Convenience and purity of sorting An FC-CS generally must be used by a number o f investigators with quite different interests. Thus it becomes important to have available a variety of sorting protocols based on what combination of signals determines which cells are to be sorted, whether yield or purity is the primary consideration or whether contaminants of a particular kind are particularly undesirable. Methods for changing from one sorting protocol to another should be rapid and simple. We have developed cell sorting electronics meeting these criteria (McCutcheon and Miller, 1981). Sorting decisions are stored in a read-only m e m o r y (ROM). All the input signals for a given event are used to define an address in this m e m o r y which contains the desired sorting o u t c o m e for the event. A set of switches enables one to choose between different sorting protocols by switching to different blocks of the m e m o r y . With properly designed control circuitry, it is possible to prevent any detected cell from being sorted along with any other detected cell if the highest purity of sorting is required. The only remaining source of cross-contamination then comes from undetected cells (the coincidence problem). It may be shown (McCutcheon, 1979) t h a t the frequency of contaminating cells in a sorted sample is given by f=

N--n

where N is the total n u m b e r of cells detected during the course of the sorting experiment, n is the number of cells detected which it is desired to sort, v' is the observed mean total cell flow rate, and r is the instrumental dead-time, the time required for processing a detected cell during which no new cells

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can be recognized. It will be seen that the fractional contamination changes by at most 2-fold when going from separating cells present in equal mixture to separating a minor c o m p o n e n t present at vanishing frequency, as long as the total cell flow rate remains constant. In our present system, the deadtime is 20 #sec and appears to be fairly typical of other systems. At a flow rate of 5000 cells/sec, up to 10% of the sorted cells will be contaminants.

Application areas (1) Surface labeling. Our procedures do not differ appreciably from those in widespread use elsewhere (see, e.g., Ledbetter and Herzenberg, 1980). We usually make a visual assessment o f fluorescence staining before sorting. However, what is seen visually and what is detected b y the photomultiplier of the sorter are n o t directly comparable. This is demonstrated in Fig. 2. The d o t t e d line shows mouse spleen cells labeled with fluoresceinated rat anti-mouse Lyt-2 h y b r i d o m a antibody. Labeled cells (peak around channel 35) comprised 12% o f the population and are well resolved from un-

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Fig. 2. Comparison of fluorescence spectra obtained for two different patterns of fluorescent staining. Solid line: nylon-wool passed murine lymph node cells incubated with fluorescein-labeled H-2-containing allogeneic membrane vesicles. Dashed line: murine spleen cells labeled with fluoresceinated rat anti-mouse-Lyt-2 hybridoma antibody.

20 labeled cells which were all below channel 3 and are n o t shown. On visual inspection, labeled cells were labeled uniformly b u t the labeled and unlabeled populations were scarcely discernible. The solid line shows the spectrum from nylon-wool passed lymph node cells which had been incubated with fluorescein-labeled, H-2-containing, allogeneic membrane vesicles (Elliott and Miller, unpublished). On visual examination, 4% of the cells b o u n d vesicles which were easily observed as discrete dots of fluorescence on the cells. However, in the fluorescence spectrum vesicle binders are scarcely resolved from vesicle non-binders. On sorting, 80% of cells above channel 12 were vesicle binders whereas less than 1% of cells below this channel were vesicle binders. In comparison with the photomultiplier, the eye is very good at detecting fluorescent cells on which the fluorescence is localized in patches b u t is rather poor at detecting fluorescent cells on which the fluorescence is uniform. It would appear that the ability of the eye to detect a fluorescent cell depends u p o n the contrast with background whereas the ability of the photomultiplier to detect a fluorescence cell depends upon the integrated intensity of the signal over the whole cell. (2) Fluorescence depolarization. The environment of a fluorescent probe can affect properties of the fluorescence detected from that probe and thus give information a b o u t that environment. Measurement of changes in fluorescence polarization is one m e t h o d for obtaining such environmental information. Operationally, this is done by exciting the fluorescence with polarized light and measuring the extent to which the emitted fluorescent radiation retains the polarization of the incoming light. If the molecules of the fluorescent probe do n o t rotate between absorption and emission, polarization will be retained; if they rotate extensively, the polarization will be totally lost. The degree of polarization of the fluorescence can be quantitated by measuring P, defined as (I1 --I2)/(I~ + I2) where I1 and I2 are, respectively, the intensities of fluorescence polarized parallel and perpendicular to the plane of polarization of the incident light. The measured polarization value depends upon both the lifetime of the fluorescent state and the rate of rotation of the fluorescent molecule, the latter being determined by the viscosity (or, inversely, fluidity) of the supporting medium with respect to the fluorescent probe. T w o main kinds of fluorescence polarization studies have been performed. In the first, lipid soluble probes such as 1,6-diphenyl-l,3,5-hexatriene have been used. These are thought to localize primarily in the lipid regions of the cytoplasmic membrane where they provide a measure of membrane fluidity (Shinitzky and Barenholz, 1978) b u t these probes can also go elsewhere (Collard and De Wildt, 1978). Using this assay, rapid changes in the membrane fluidity of T l y m p h o c y t e s can be detected after exposure to certain lymphokines (Puri et al., 1980). In the second kind of study, one places viable cells in medium containing fluorescein diacetate (FDA) which is itself non-fluorescent. Once in the cytoplasm, the FDA undergoes enzymatic hy-

21 drolysis into free fluorescein which is fluorescent. In viable cells, FDA can enter a cell more readily than fluorescein can leave, leading to the accumulation of fluorescein in the cytoplasm, a p h e n o m e n o n called fluorochromasia (Rotman and Papermaster, 1966). To what extent the fluorescein is uniformly distributed throughout the aqueous phase of the cytoplasm, b o u n d to cytoplasmic proteins or segregated in specific subcellular organelles is n o t clear (Cercek and Cercek, 1979; Meisingset and Steen, 1981). Most studies of fluorescence polarization have used a fluorimeter. This has a n u m b e r of disadvantages. First, for those using FDA, fluorescein leaks o u t of the cells rather rapidly ( R o t m a n and Papermaster, 1966), necessitating extensive correction of the data to distinguish between the polarization measured for fluorescein molecules inside and outside the cells being analyzed. Second, the signal can become depolarized by scatter events occurring before the signal is analyzed. Third, for a heterogeneous cell sample, the value obtained will be weighted by those cells containing the most fluorescent material. Use of an FC-CS avoids all of these problems. In addition, one measures P values of individual cells rather than an average value for a whole population enabling one potentially to identify cell subpopulations differing in P value. Note also that an argon ion laser beam is usually highly polarized as produced. Our experience is restricted to studies using FDA. It has been shown for several different stimuli that when the stimulus is added to a heterogeneous cell suspension, those cells responding to the stimulus can show a change in polarization within 30 min of adding the stimulus. Colonies of T cells can be grown from human peripheral blood (Price et al., 1978, 1980a). Growth of the colonies requires addition of a factor (or factors) derived from the supernatants of human blood leukocytes cultured with phytohemagglutinin. Addition of the supernatant to fresh peripheral blood l y m p h o c y t e s causes a large (at least 20%) reduction in the fluorescence polarization of fluorescein molecules in their cytoplasm (Price et al., 1978). When cells showing the largest downshift are sorted, they are greatly enriched in their ability to form T colonies (Price et al., 1978). Erythropoietin-responsive cells (Krosgrud, Stewart, Van-Zant, Goldwasser and Price, manuscript in preparation) and granulopoietic progenitor cells from bone marrow (Price and Krogsrud, 1978; Price et al., 1980b) can also be sorted in the same way. It appears that lymphocytes from a cancer patient can also be distinguished from those of a normal control in a similar way (Cercek et al., 1974; Cercek and Cercek, 1977; Stewart et al., 1979). When the patient's l y m p h o c y t e s are incubated with an extract of the tumor, they usually undergo a downshift in polarization whereas control l y m p h o c y t e s or l y m p h o c y t e s from a patient with an unrelated t u m o r do not. (3) Use of Hoechst 33342 dye. The bisbenzimidazole dye, Hoechst 3 3 3 4 2 (HO342) (Loewe and Urbanietz, 1974) has at least 3 areas of application: (i) sorting of cells on the basis of DNA content; (ii) sorting of activated T cells from other T cells; and (iii) sorting of l y m p h o c y t e subpopulations.

22 The dye binds both specifically and quantitatively to DNA and, once bound, becomes strongly fluorescent (Latt and Stetten, 1976). Unlike other DNAspecific fluorescent dyes (Crissman et al., 1979), H O 3 4 2 is readily taken up by living cells and is non-toxic (Arndt-Jovin and Jovin, 1977). Thus, it can be used to sort viable cells on the basis of DNA content and the sorted cells can be tested in functional assays. We have successfully performed such studies (Pallavicini et al., 1979). The other applications of the dye arise from our observation (Lalande and Miller, 1979) that, under appropriate staining conditions, H 0 3 4 2 stained murine l y m p h o c y t e populations show differences in fluorescence intensity n o t related to DNA content. We have shown that the dye gets into a living cell via an unmediated diffusion process and that the rate at which the DNA becomes stained is determined by the rate at which the dye is transported across the cytoplasmic membrane (Lalande et al., 1981). When murine lymph node (LN) cells are incubated in the dye, t w o populations of lymphocytes are seen which we have called LI (low intensity) and HI (high intensity). Loken (1980) has shown that the LI cells are predominantly T cells and the HI cells B cells. We found (Lalande and Miller, 1979) that on stimulating LN cells with the mitogen, concanavalin A (Con A), a substantial fraction of the T cells moved from the LI to the HI peak. This shift started becoming detectable 3 h after adding the Con A. In a mixed l y m p h o c y t e reaction, a much smaller number of T cells move from the LI to HI peak. Among these are c y t o t o x i c T l y m p h o c y t e precursor cells specific for the stimulator cells of the mixed l y m p h o c y t e reaction, all other c y t o t o x i c lymp h o c y t e precursor cells remaining in the LI peak (Lalande et al., 1980). We conclude that an increase in membrane permeability is an early event in T l y m p h o c y t e activation and that this change can be detected using H O 3 4 2 dye. Whether activation of cell types other than T cells can also be detected is n o t known at the present time. We also conclude that resting B and T cells differ in their membrane permeability and that this difference allows them to be separated following staining with HO342 dye. Preliminary examination of thymus and bone marrow indicates that several other subpopulations may be detectable in this way (Lalande, unpublished). Although technical details are described fully elsewhere (Lalande et al., 1979, 1980, 1981), we will make a few comments here. If cells are incubated in H O 3 4 2 dye for a sufficiently long time, the amount of fluorescence seen will be directly proportional to the a m o u n t of DNA. For LN l y m p h o c y t e s incubated in 2.5 pM dye at 37°C in serum-free tissue culture medium, this time is a b o u t 1.5 h whereas, for the same conditions, an incubation time of 10 rain procedures o p t i m u m separation of the LI and HI peaks. These times are an order of magnitude longer if serum is present in the incubation medium. Dye neither gets into or gets o u t of cells at 4 ° C. It is very difficult to wash all the d y e from the cells, even at 37°C. However, even if first washed, dye can escape from labeled cells incubated at 37°C and be picked up by previously unlabeled cells. The dye is slightly toxic so that toxicity

23

controls should be included in any experiment. The optimum wavelength for exciting fluorescence from the dye is in the U.V. We use the combined 351 and 364 nm lines from an argon ion laser selected for good U.V. output {model 164-05, Spectra-Physics Inc., Mountain View, CA). An output of 50 mW is sufficient. REFERENCES Arndt-Jovin, D.J. and T.M. Jovin, 1977, J. Histochem. Cytochem. 25,585. Arndt-Jovin, D.J. and T.M. Jovin, 1978, Ann. Rev. Biophys. Bioeng. 7,527. Bonnet, W.A., H.R. Hulett, R.G. Sweet and L.A. Herzenberg, 1972, Rev. Sci. Instrum. 43,404. Cercek, L. and B. Cercek, 1977, Eur. J. Cancer 13,903. Cercek, L. and B. Cercek, 1979, Biophys. J. 28,403. Cercek, L., B. Cercek and C.I.V. Franklin, 1974, Br. J. Cancer 29,345. Collard, J.G. and A. De Wildt, 1978, Exp. Cell Res. 116,447. Crissman, H.A., P.F. Mullaney and J.A. Steinkamp, 1975, in: Methods in Cell Biology, Vol. IX, ed. D.A. Prescott (Academic Press, New York) p. 179. Crissman, H.A., A.P. Stevenson, R.J. Kissane and R.A. Tobey, 1979, in: Flow Cytometry and Sorting, eds. M.R. Melamed, P.F. Mullaney and M.L. Mendelsohn (Wiley, New York) p. 243. Horan, P.K. and L.L. Wheeless, 1977, Science 198,149. Lalande, M.E. and R.G. Miller, 1979, J. Histochem. Cytochem. 27,394. Lalande, M.E., M.J. McCutcheon and R.G. Miller, 1980, J. Exp. Med. 151, 12. Lalande, M.E., V. Ling and R.G. Miller, 1981, Proc. Natl. Acad. Sci. U.S.A. 78,363. Latt, S.A. and G. Stetten, 1976, J. Histochem. Cytochem. 24, 24. Ledbetter, J.A. and L.A. Herzenberg, 1980, Immunol. Rev. 47, 63. Lindmo, T. and H.B. Steen, 1977, Biophys. J. 18,173. Loewe, H. and J. Urbanietz, 1974, Arzneim. Forsch. 24, 1927. Loken, M.R., 1980, J. Histochem. Cytochem. 28, 36. Loken, M.R. and L.A. Herzenberg, 1975, Ann. N.Y. Acad. Sci. 254,263. McCutcheon, M.J., 1979, M.Sc. Thesis, University of Toronto. McCutcheon, M.J. and R.G. Miller, 1979, J. Histochem. Cytochem. 27,246. McCutcheon, M.J. and R.G. Miller, 1981, Cytometry, in press. Meisingset, K.K. and H.B. Steen, 1981, Cytometry 1,272. Miller, R.G., 1977, Immunol. Ser. 5,205. Miller, R.G. and R.A. Phillips, 1969, J. Cell. Physiol. 73,191. Miller, R.G. and G.B. Price, 1979, Clin. Hematol. 8,421. Miller, R.G., R.M. Gorczynski, L. Lafleur, H.R. MacDonald and R.A. Phillips, 1975, Transplant. Rev. 25, 59. Pallavicini, M.G., M.E. Lalande, R.G. Miller and R.P. Hill, 1979, Cancer Res. 39, 1891. Price, G.B. and R.L. Krogsrud, 1978, in: Differentiation of Normal and Neoplastic Hematopoietic Cells (Cold Spring Harbor Laboratories, New York) p. 371. Price, G.B., M.J. McCutcheon, W.B. Taylor and R.G. Miller, 1977, J. Histochem. Cytochem. 25,597. Price, G.B., R. Krogsrud, S. Stewart and J.S. Senn, 1978, in: Hematopoietic Cell Differentiation (Academic Press, New York) p. 417. Price, G.B., S. Stewart and R.L. Krogsrud, 1979, Blood Cells 5,161. Price, G.B., H.-S. Teh and R.G. Miller, 1980a, J. Immunol. 124, 2352. Price, G.B., C.J. O'Hara, R. Krogsrud and S.S. Stewart, 1980b, Blood Cells 6,689. Puri, J., M. Shinitzky and P. Lonai, 1980, J. Immunol. 124, 1937. Rotman, B. and B.W. Papermaster, 1966, Proc. Natl. Acad. Sci. U.S.A. 55, 134. Shinitzky, M. and Y. Barenholz, 1978, Biochim. Biophys. Acta 515,367.

24 Shortman, K., 1972, Ann. Rev. Biophys. Bioeng. 1, 93. Shortman, K., N. Williams and P. Adams, 1972, J. Immunol. Methods 1,273. Stewart, S.S., K.I. Pritchard, J.W. Meakin and G.B. Price, 1979, Clin. Immunol. Immunopathol. 13, 171. Stewart, S.S., R.G. Miller and R.G. Price, 1980, Cytometry 1, 2D4.