Simultaneous flow cytometric measurement of viability and lymphocyte subset proliferation

Journal of Immunological Methods 247 (2001) 175–186 www.elsevier.nl / locate / jim

Simultaneous flow cytometric measurement of viability and lymphocyte subset proliferation a, a a b Ingrid Schmid *, Mary Ann Hausner , Steve W. Cole , Christel H. Uittenbogaart , Janis V. Giorgi a , Beth D. Jamieson a b

a Department of Hematology /Oncology, UCLA School of Medicine, 12 -236 Factor Building, Los Angeles, CA 90095, USA Department of Microbiology and Immunology, and Department of Pedriatics, UCLA School of Medicine, Los Angeles, CA 90095, USA

Received 15 August 2000; accepted 23 October 2000

Abstract Combined analysis of DNA content and immunofluorescence on single cells by flow cytometry provides information on the proliferative response of cellular sub-populations in mixed cell preparations. However, the presence of considerable numbers of dead (nonviable) cells impairs accurate flow cytometric data analysis, mainly, because dead cells can bind antibodies non-specifically and show alterations in their DNA staining profiles. We developed a rapid method for identification of dead cells by fluorescence in cell preparations that are stained simultaneously for two-color immunofluorescence and DNA content. Cells are stained with 7-aminoactinomycin D (7-AAD) for dead cell discrimination and with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE)-labeled monoclonal antibodies (mAb) for cell surface immunofluorescence. Diffusion of 7-AAD from stained, dead cells into unstained, live cells after cell permeabilization is blocked by the addition of its non-fluorescent analogue actinomycin D (AD). DNA is stained with red-excitable TO-PRO-3 iodide (TP3) which has an emission spectrum that can be effectively separated from the emissions of FITC, PE, and 7-AAD. TP3 staining is performed in the presence of ribonuclease A (RNAse) in phosphate–citrate buffer containing saponin (PCBS) at low pH. FITC fluorescence is sensitive to acid pH; therefore, PCBS is replaced after DNA staining with 13 PBS at pH 7.2 containing saponin to permit accurate detection of FITC immunofluorescence on the flow cytometer. We apply this method to the analysis of differential proliferation of lymphocyte subsets in cultures of human peripheral blood mononuclear cells (PBMC) with low viability.  2001 Elsevier Science B.V. All rights reserved. Keywords: Flow cytometry; Cell death; Permeabilization; Dead cell exclusion; Immunofluorescence; DNA content; Proliferation; CD3 / CD28 stimulation; 7-Aminoactinomycin D; TO-PRO-3

Abbreviations: 7-AAD, 7-aminoactinomycin D; AD, actinomycin D; BP, band pass filter; CV, coefficient of variation; FITC, fluoresceinisothiocyanate; mAb, monoclonal antibody; NaAz, sodium azide; NCS, newborn calf serum; PBMC, peripheral blood mononuclear cells; PCB, phosphate–citrate buffer; PE, phycoerythrin; PI, propidium iodide; RNAse, ribonuclease A; TP3, TO-PRO-3 iodide *Corresponding author. Tel.: 11-310-206-7289; fax: 11-310-794-2145. E-mail address: [email protected] (I. Schmid). 0022-1759 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0022-1759( 00 )00323-9

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1. Introduction Simultaneous flow cytometric assessment of phenotype and DNA content is an established method to measure subset proliferation or arrest in mixed cell preparations without the need for prior cell separation (Loken, 1980; Kruth et al., 1981; Rabinovitch et al., 1986; Schmid et al., 1991, 2000a; Storek et al., 1992). However, whenever considerable numbers of dying or dead cells are present data analysis can become difficult. Dead cells can compromise the accuracy of flow cytometric analysis, primarily due to their increased autofluorescence (Zamai et al., 1993), nonspecific binding of monoclonal antibodies (Terstappen et al., 1988; Riedy et al., 1991; Stewart and Stewart, 1994), altered expression of cell surface antigens (Schmid et al., 1992, 1994a,b) and DNA content (Muirhead et al., 1985; Darzynkiewicz et al., 1992). Dead (nonviable) cells can be identified on the flow cytometer by changes in their light scatter properties — they generally show a decrease in forward scatter and an increase in side scatter as compared to live cells. However, dead cell discrimination by light scatter is often unreliable (Dangl et al., 1982); this is particularly notable in heterogeneous samples and in cell preparations that were fixed / permeabilized for DNA staining. In both cases, the scatter distinction between live and dead cells is frequently insufficient for accurate dead cell exclusion (Penttila et al., 1975; Muirhead et al., 1985; Schmid et al., 1999). Flow cytometric detection of dead cells can be enhanced by addition of various fluorescent non-vital DNA dyes that enter cells which have lost membrane integrity (Dangl et al., 1982; Bohmer, 1985; Schmid et al., 1992). One of these DNA dyes, 7-aminoactinomycin D (7-AAD), can also be used with cells that are permeabilized subsequent to 7-AAD staining, because its diffusion from stained dead cells into unstained live cells after cell fixation and / or permeabilization is prevented by addition of its nonfluorescent analogue actinomycin D (AD) (Fetterhoff et al., 1993; Schmid and Giorgi, 1995). We have shown recently that 7-AAD can be used for dead cell exclusion in cell preparations that are simultaneously stained for DNA content using pyronin Y(G) (PY) and single-color immunofluorescence using fluorescein-isothiocyanate (FITC)-labeled antibodies (Schmid et al., 1999; Schmid, 1999). In our pub-

lished protocol, PY is utilized for DNA staining, because propidium iodide (PI), the most commonly used dye for DNA content measurements by flow cytometry, cannot be combined with 7-AAD due to its extensive spectral emission overlap with 7-AAD. However, PY DNA staining is problematic, because commercially available preparations of PY can differ in purity and water content (Schmid, 1999). Furthermore, PY is a nonspecific polyanion nucleic acid stain which binds preferentially to RNA (Shapiro, 1981, 1995) and yields disappointingly broad DNA peaks even in the presence of high concentrations of RNAse (Schmid et al., 1999; Schmid, 1999). Therefore, we searched for an alternate DNA dye that produces DNA histograms with low coefficients of variation (CV) and is compatible with 7-AAD staining. TO-PRO-3 iodide (TP3) is a nucleic acidspecific dye that can be excited by a red-emitting diode laser (Doornbos et al., 1994) as available on our four-color, dual-laser benchtop flow cytometer. It has been demonstrated previously that by using this excitation light source TP3 DNA histograms with low CVs can be generated (Doornbos et al., 1994) and that the far red emission from TP3 can be effectively separated from the emissions of FITC and PE, respectively (Van Hooijdonk et al., 1994). Here we show that TP3 DNA staining can be combined with 7-AAD viability staining and with simultaneous detection of two cell surface antigens using FITC and PE-labeled monoclonal antibodies. 7-AAD staining of dead cells is maintained during subsequent procedural steps by addition of nonfluorescent AD. DNA staining is performed in the presence of RNAse in phosphate–citrate buffer at low pH containing saponin (PCBS) with TP3 at a non-saturating concentration. PCBS is replaced with 13 PBS at pH 7.2 containing saponin before sample acquisition on the flow cytometer to restore FITC fluorescence which is dramatically reduced by acid pH. The improved flow cytometry technique presented here permits reliable dead cell exclusion in cell preparations with low viability for accurate correlated subset and DNA content analysis. 2. Materials and methods

2.1. Cell lines and culture conditions Molt-4f, a T-cell leukemia line, was maintained in

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continuous culture in serum-free medium as described previously (Uittenbogaart et al., 1983). On occasion, cells were grown under sub-optimal conditions to obtain cell preparations that contained considerable numbers of dying and dead cells. CEM, a second T-cell leukemia line, was maintained in continuous culture in RPMI 1640 (Life Technologies, Rockville, MD, USA) supplemented with 10% fetal bovine serum (Omega Scientific, Tarzana, CA, USA), 100 U / ml penicillin, 100 mg / ml of streptomycin, and 2 mM glutamine (all from Life Technologies). Healthy donor peripheral blood mononuclear cells (PBMC) were obtained from commercial leukopaks and separated by Ficoll density gradient essentially ¨ as described by Boyum (1968). PBMC were washed twice with PBS and were stained immediately or were cultured for 3, 4, or 5 days either in medium alone (RPMI 1640, supplemented with 10% (v / v) human AB serum (Sigma, St. Louis, MO, USA), 100 U / ml penicillin, 100 mg / ml of streptomycin, and 2 mM glutamine) or in this medium containing 100 ng / ml of soluble CD28.2 (Biodesign, Kennebumkport, ME, USA) in the presence of 100 ng / ml of plate-bound OKT3 (Ortho Diagnostics, Raritan, NJ, USA). Cultured PBMC were washed once in PBS.

2.2. Reagents and monoclonal antibodies Phosphate buffered saline (PBS without Ca 21 and Mg 21 ) and newborn calf serum (NCS) were purchased from Irvine Scientific (Irvine, CA, USA). Sodium azide (NaAz), phosphate–citrate buffer tablets, sodium chloride, sodium ethylene-diaminetetraacetic acid (EDTA), bovine serum albumin (BSA), ribonuclease A (RNAse), dimethylsulfoxide (DMSO), and saponin were from Sigma. Fluorescein-isothiocyanate (FITC)-labeled CD8, phycoerythrin (PE)-labeled CD4, FITC and PE-labeled mouse IgG 1 isotypic controls were all obtained from Becton Dickinson Biosciences (San Jose, CA, USA). CD5 FITC and CD8 PE were from Caltag Laboratories (Burlingame, CA, USA). Propidium iodide (PI) and 7-aminoactinomycin D (7-AAD) were purchased from Calbiochem (San Diego, CA, USA). TO-PRO3 iodide (TP3) was from Molecular Probes (Eugene, OR, USA). Actinomycin D (C 1 ) (AD) was obtained from Roche Molecular Biosystems (Indianapolis, IN, USA). An AD stock solution was prepared by

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dissolving 1 mg of AD powder first in 50 ml of DMSO, then adding 950 ml of 13 PBS.

2.3. Staining of dead cells and cell surface antigens PBS-washed cells (1310 6 ) were placed into a 12375 mm tube. Then, 250 ml of PBS supplemented with 2% NCS and 0.1% NaAz and containing 4 mg / ml of 7-AAD were added and mixed well. For experiments that tested various concentrations of 7-AAD the amount of 7-AAD indicated in the text was added. When cells were stained simultaneously for surface antigen expression, appropriate amounts of FITC-labeled and PE-labeled mAb or of FITC and PE isotypic control antibody (20 ml for mAbs or control antibodies from BD Biosciences, 5 ml for mAbs from Caltag) were added at this time. Cells were then incubated while protected from light for 15 min at 20–258C. Next, cells were washed once with 2 ml of 13 PBS by centrifugation at 2503g for 5 min. The supernatant was removed completely and 2 ml of 13 PBS containing 4 mg / ml of AD (PBS / AD) were added. The mixture was vortexed immediately, centrifuged for at least 5 min at 2503g, and the supernatant was removed completely.

2.4. DNA staining using TO-PRO-3 and propidium iodide PBS-washed cells (1310 6 ) or cells stained with 7-AAD or cells costained with 7-AAD and mAbs were resuspended in 0.5 ml of phosphate–citrate buffer solution (0.1 M phosphate–citrate, 0.15 M of NaCl, 5 mM sodium EDTA, 0.5% BSA, pH 4.8) containing 0.02% of saponin (PCBS), 4 mg / ml of AD, 0.5 mM of TP3, and 200 mg / ml of RNAse followed by incubation for 30 min at 20–258C. For experiments that tested DNA staining using different TP3 concentrations, the amount of TP3 as indicated in the text was added. For propidium iodide (PI) DNA staining, cells were resuspended in PCBS containing 10 mg / ml of PI and 200 mg / ml of RNAse followed by incubation for 30 min at 20–258C. For cells stained for cell surface immunofluorescence with FITC-labeled mAbs, cells were spun down after DNA staining by centrifugation at 2503g for 5 min; then, the cell pellet was resuspended in 0.5 ml of 13 PBS at pH 7.2 containing 0.02% of saponin, 0.5 mM

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of TP3, and 4 mg / ml of AD to restore the FITC fluorescence that is markedly diminished at pH 4.8. All samples were acquired on the flow cytometer in their staining solutions.

2.5. Flow cytometry Samples were analyzed on a FACSCalibur flow cytometer (BD Biosciences) equipped with a 15 mW air-cooled 488 nm argon-ion laser for excitation of FITC, PE, and 7-AAD and a 635 nm red diode laser for excitation of TP3. Green FITC fluorescence was collected after a 530 / 30 nm band pass (BP) filter. Orange emission from PE and from PI were filtered through a 585 / 42 nm BP filter. Red TP3 emission was collected through a 661 / 16 BP filter and far red 7-AAD emission through a 670 longpass filter. Electronic compensation was used among the fluorescence channels to remove residual spectral overlap. Photomultiplier tube voltage and spectral compensation were initially set using cells single-stained with FITC alone, PE alone, TP3 alone, or 7-AAD alone. Settings on the flow cytometer were then further optimized in samples stained with combinations of the different fluorochromes to compensate for their interactions. FITC, PE, and 7-AAD fluorescence data were all displayed on four-decade log scales. PI and TP3 fluorescence were both collected using linear amplification. For acquisition of TP3stained cells, in addition to FL-4 (TP3 fluorescence) height (H), FL-4 area (A) and width (W) were measured for doublet discrimination. For acquisition of PI-stained samples, FL-2 (PI fluorescence) H, FL-2 A and FL-2 W were collected for doublet discrimination. The low (12 ml / min) or intermediate (35 ml / min) flow rate setting was used for sample acquisition to improve the coefficient of variation on DNA histograms. A minimum of 10,000 events was collected on each sample. Analysis of the multivariate data was performed with CELLQuestE software (BD Biosciences). Cell cycle analysis of DNA histograms was performed with ModFit LT E software (Verity Software House, Topsham, ME, USA). 3. Results and discussion

3.1. Optimization of 7 -AAD dead cell staining We have recently shown that using phosphate–

citrate buffer at pH 4.8 containing saponin (PCBS) for cell membrane permeabilization provides excellent nucleic acid staining conditions while preserving cell surface immunofluorescence (Schmid et al., 2000a,b). Therefore, we wanted to combine this simple and rapid DNA staining protocol with dead cell exclusion. However, in contrast to several 7AAD dead cell discrimination techniques published earlier (Fetterhoff et al., 1993; Schmid et al., 1994b, 1999; Schmid, 1999), the current method does not include cell fixation with formaldehyde; therefore, it was necessary to find out if 7-AAD-stained dead cells could be sufficiently discriminated from live cells after cell permeabilization with PCBS. Fig. 1 shows one representative experiment of three where we investigated dead cell discrimination using various 7-AAD staining concentrations in combination with PCBS. Numbers of dead cells with lost membrane integrity were very comparable with all three staining concentrations of 7-AAD we tested. After cell permeabilization with PCBS (Fig. 1, row B), staining intensity of the 7-AAD 1 cells decreased markedly, but live cell frequencies remained similar and continued to correlate with cell viability as determined by trypan blue exclusion. Based on these results we decided to continue our experiments with 4 mg / ml of 7-AAD, the same dye concentration we had used previously for staining of dead cells in fixed and permeabilized cell preparations (Schmid et al., 1999; Schmid, 1999) and with 4 mg / ml of AD for prevention of dye transfer to permeabilized live cells.

3.2. Titration of TP3 DNA staining TO-PRO-3 iodide (TP3) is a newer monomeric cyanine nucleic acid dye suitable for multi-color staining using FITC and PE (Van Hooijdonk et al., 1994). TP3 intercalates into double-stranded nucleic acids with a preference for AT base pairs and with a large fluorescence enhancement as compared to the fluorescence of unbound dye. It can be excited either with a helium neon laser (Van Hooijdonk et al., 1994) or a red diode laser (Doornbos et al., 1994) and has an emission peak with a maximum around 661 nm, however it also emits considerable fluorescence in FL-3 (7-AAD channel) (Tom Frey, personal communication, February 1, 1999 and data not shown). Therefore, we investigated the possibility of

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Fig. 1. Molt-4f cells (viability by trypan blue exclusion: 70%) were stained with 7-AAD at the concentration (in mg / ml) indicated in the contour plots without (A) or with subsequent permeabilization with PCBS (B) as otherwise described in the Methods with a concentration of AD in the washing solutions and in PCBS that matched the concentration of 7-AAD for staining. All contour plots show the percentages of live, 7-AAD-negative cells.

lowering the staining concentration of TP3 to minimize the FL-4 signal in FL-3 and to limit the interference fluorescent dyes in solution can exert on other fluorochromes either by energy transfer or by fluorescence quenching (Telford et al., 1994; Stokke et al., 1998; Schmid et al., 1999; Schmid, 1999). Table 1 shows one representative experiment of four individual experiments. Data indicate that DNA content distributions of CEM cells remained similar with TP3 concentrations from 10 to 0.1 mM and compared well with data obtained with propidium iodide staining. However, with the exception of 0.1 mM TP3, concentrations at or below 3 mM TP3 decreased the CVs of the G 1 peaks markedly thus improving the precision of DNA measurements. Furthermore, as reported earlier by Van Hooijdonk et al. (1994), high TP3 concentrations resulted in lower staining intensities as determined by the position of the G 1 peak on the fluorescent scale, possibly due to interference of the blue color of the dye with the measurement. The presence of AD in solution is

necessary for maintenance of 7-AAD dead cell staining when cells are permeabilized, therefore we also wanted to test if AD would adversely affect TP3 DNA content measurements. To this end we compared cell cycle data obtained without AD with data measured when 20 mg / ml AD was added to PCBS. As seen in Table 1, both data sets compared well, even in the presence of high concentrations of AD. Based on data shown in Table 1 we decided that we could use the subequilibrium concentration of 0.5 mM TP3 for subsequent experiments to reduce TP3 fluorescence in FL-3 and minimize the interaction of TP3 with the other fluorochromes we wanted to measure.

3.3. Combination of 7 -AAD and TP3 staining Next, we investigated the ability of 7-AAD to provide improved discrimination of dead cells in samples stained with TP3 for DNA content. As previously observed (Schmid, 1999) and as seen in

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Table 1 Effects on DNA content analysis of TP3 staining concentration and addition of AD Dye (conc.)

Cell cycle distribution %G 1

%S

%G 21M

%CV G 1

G 1 peak position

TP3 (10 mM) TP3 (5 mM) TP3 (3 mM) TP3 (1 mM) TP3 (0.5 mM) TP3 (0.2 mM) TP3 (0.1 mM) PI (10 mg / ml)

36 37 35 38 37 38 35 37

62 60 58 54 55 55 57 58

2 5 8 8 8 7 8 5

7.1 5.6 4.9 4.4 4.3 4.8 6.9 5.7

79 113 119 83 54 29 18

TP3 (10 mM)1AD a TP3 (5 mM)1AD a TP3 (3 mM)1AD a TP3 (1 mM)1AD a TP3 (0.5 mM)1AD a TP3 (0.2 mM)1AD a TP3 (0.1 mM)1AD a

36 38 39 40 40 40 36

57 53 53 51 54 53 63

7 9 8 9 6 7 1

6.0 5.5 5.3 4.5 4.0 4.4 7.9

a

In each sample, DNA staining with TP3 on CEM cells was performed in the presence of 20 mg / ml of AD.

Fig. 2 on Molt-4f cells permeabilized with PCBS, scatter discrimination between live and dead cells is not feasible due to considerable overlap of their scatter profiles. In contrast, 7-AAD-negative, live cells form a distinct cluster on the TP3 versus 7AAD fluorescence contour plot and thus permit reliable live cell analysis. Cell cycle distribution between all cells and live cells differed markedly; 7-AAD-negative cells showed a higher proportion of cells in G 1 and fewer cells in the S and G 21M phases of the cell cycle, indicating that cells in proliferative states had died preferentially. Furthermore, the CV of the G 1 peak decreased when only live cells were analyzed. Data shown in Fig. 2 are representative of three separate experiments, correlate with results we obtained by using 7-AAD for dead cell exclusion and DNA PY staining (Schmid et al., 1999), and emphasize the need for dead cell exclusion in cell preparations with low viability.

3.4. Viability, dual-color immunofluorescence, and DNA content To test the applicability of our method to the immune subset analysis in human lymphocytes, we performed the protocol as described in the Methods

on freshly isolated human peripheral blood mononuclear cells (PBMC) that were heated to 558C for 10 min to increase the numbers of dead cells in the cell preparation. Previously, when we used PCBS cell permeabilization at pH 4.8 in combination with cell surface immunofluorescence (Schmid et al., 2000a,b) we resorted to the pH insensitive, green-emitting fluorochrome Alexa Fluor 488E instead of FITC. However, monoclonal antibodies directly conjugated to Alexa Fluor 488E are not as readily commercially available as FITC-labeled antibodies. FITC fluorescence is not permanently lost at low pH; therefore, we tested the feasibility of replacing PCBS after DNA staining with PBS at pH 7.2 for detection of FITC immunofluorescence without causing adverse effects on DNA content measurements. As seen in Fig. 3, detection of CD8 1 lymphocytes stained with CD8-FITC was compromised when cells were analyzed on the flow cytometer in PCBS at pH 4.8 while CD4 PE was unaffected (Fig. 3C). However, when samples were resuspended in PBS at neutral pH before acquisition, CD8 1 and CD4 1 lymphocyte frequencies in cells stained simultaneously with 7AAD and TP3 (Fig. 3E) closely matched the percentages of these subsets in unpermeabilized cells single-stained with either CD8 FITC (Fig. 3A) or

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Fig. 2. Molt-4f cells (viability by trypan blue exclusion: 68%) were stained with 7-AAD and with TP3 as described in the Methods. (A) Forward vs. side scatter contour plot, ungated. (B) TP3 fluorescence vs. 7-AAD fluorescence contour plot, ungated. (C) Histogram of TP3 fluorescence, gated on FL-4 width (W) vs. FL-4 area (A) for doublet discrimination. (D) Histogram of TP3 fluorescence of cells within R1 as indicated in (B), gated on FL-4 W vs. FL-4 A for doublet discrimination.

CD4 PE (Fig. 3B), respectively. Furthermore, the CV of the G 0 peak of the TP3 DNA histogram remained low (Fig. 3F).

3.5. Live lymphocyte subset proliferation during CD3 /CD28 costimulation Fig. 4 presents the application of the current method to the analysis of human PBMC that were CD3 / CD28 costimulated for 3, 4, and 5 days without change of culture medium and then stained with 7-AAD, CD5 FITC, CD8 PE, and with TP3 as described in the Methods. One representative experiment of four is shown. Remarkably, dead cells were not visible on any scatter contour plot, but could be easily discriminated by their up-take of 7-AAD. The frequency of dead, 7-AAD 1 cells remained very low

through day 3; however, on day 4, 29% of the cells had lost membrane integrity and on day 5 the percentage of dead cells had increased to 53%. As expected with this T-cell stimulation system (June et al., 1990), by day 3 of CD3 / CD28 costimulation 71% of CD8 1 cells which express the T lymphocyte antigen CD5 were in the S and G 21M phases of the cell cycle; the frequency of proliferating cells remained similar through day 4, but on day 5 it started to decrease slightly, suggesting that CD8 1 CD5 1 cells were past peak proliferation. CD8 1 cells which do not coexpress CD5 (NK cells) also proliferated, but to a much lesser degree than CD8 1 T cells. Furthermore, the proportion of cells in proliferative states already decreased on day 4 as compared to day 3, and by day 5 most CD8 1 CD5-negative cells were in G 0 / 1 . These data show that in cultures stimulated

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Fig. 3. Freshly isolated human PBMC were heated to 558C for 10 min. Cells were single-stained with either CD8 FITC or CD4 PE without permeabilization (A,B). Cells were cell surface stained with CD8 FITC and CD4 PE in the presence of 7-AAD, permeabilized and stained with TP3 in PCBS as described in the Methods, then analyzed on the flow cytometer in PCBS pH 4.8 (C) or analyzed on the flow cytometer in 13 PBS pH 7.2 as described in the Methods (D–F). (A) Histogram of CD8 FITC fluorescence, gated on lymphocytes. (B) Histogram of CD4 PE fluorescence, gated on lymphocytes. (C) Contour plot of CD8 FITC fluorescence vs. CD4 PE fluorescence, gated on lymphocytes. (D) Forward scatter vs. 7-AAD fluorescence contour plot, gated on lymphocytes. (E) Contour plot of CD8 FITC fluorescence vs. CD4 PE fluorescence, gated on lymphocytes and 7-AAD-negative cells as shown in (D). (F) Histogram of TP3 fluorescence, gated on lymphocytes and 7-AAD-negative cells as shown in (D) and gated on FL-4 width vs. FL-4 area for doublet discrimination. Histogram and contour plot cursor positions for determination of positive cell frequencies were set according to the appropriate isotype control background levels.

with CD3 and CD28.2 CD8 1 non-T-cell proliferation differs in intensity and time course from CD8 1 T-cell proliferation. Our results concur with our earlier data on CD8 / CD5 subset proliferation (Schmid et al., 2000b) that indicated that a subset of CD8 1 non-T cells reacts with a delayed time course to costimulation with CD3 / CD28, possibly due to by-stander stimulation with cytokines that T cells are known to produce in large quantities upon activation (Cole et al., 1998). PBMC cultured in parallel without CD3 / CD28 costimulation did not proliferate and did not show increased cell death during the entire culture period of 5 days (data not shown). Thus, the current method permits accurate measurement of the activation time course of specific live

lymphocyte subsets in culture systems where cell proliferation is accompanied by massive cell death.

4. Summary We present here an improved method for simultaneous detection of cell viability, two cell surface antigens, and DNA content using a benchtop duallaser flow cytometer. We show that using a simple and rapid cell preparation method for DNA content analysis dead cells can be clearly identified in heterogeneous, permeabilized cell preparations and thus can be excluded for increased accuracy in data collection. Our data indicate that reliable cell cycle

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Fig. 4. Human PBMC were cultured in the presence of CD3 and CD28.2 as described in the Methods. Column 1: Forward vs. side scatter contour plots, ungated. Column 2: Forward scatter vs. 7-AAD fluorescence contour plots with the percentage of live, 7-AAD-negative cells falling within R2 indicated; except for the day 0 contour plot which is gated on lymphocytes within R1, contour plots for all other time points are ungated. Column 3: Contour plots of CD5 FITC fluorescence vs. CD8 PE fluorescence, gated for each time point on live cells within R2 as shown in column 2; frequencies of lymphocyte subsets as shown were determined initially by setting quadrant cursor positions according to isotype control background levels, then, for cultured cells, they were optimized to delineate the division between positive and negative cell clusters (Keeney et al., 1998). Column 4: Histograms of TP3 fluorescence, gated for each time point on live cells within R2 as shown in column 2, on gate R3 for CD8 1 CD5-negative cells as shown in column 3, and on FL-4 width (W) vs. FL-4 area (A) for doublet discrimination. Column 5: Histograms of TP3 fluorescence, gated for each time point on live cells within R2 as shown in column 2, on gate R4 for CD8 1 CD5 1 cells as shown in column 3, and on FL-4 W vs. FL-4 A for doublet discrimination.

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Fig. 4. (continued)

measurements with low CV can be obtained by using TP3 DNA staining. In addition, our method permits clear resolution of dual-color immunofluorescently stained cells from background. It has been applied to the measurement of live cell proliferative responses to a T-cell stimulus of lymphocyte subsets as iden-

tified by two cell surface markers. Potential extension of this technique to the investigation of other cell types and culture systems, in particular longterm cultures with low viability, will facilitate the collection of biologically relevant data despite extensive cell death.

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Acknowledgements This work was performed in the UCLA Jonsson Comprehensive Cancer Center and Center for AIDS Research Flow Cytometry Core Facility and was supported by National Institutes of Health awards CA-16042, AI-28697, HD-29341, and HD-37597. We thank the late Dr. Janis Giorgi for her encouragement to undertake this project, Deidre ScriptureAdams for helpful discussions, Deborah AnismanPosner, Silvia Neagos, and Roger Shih for excellent assistance with cell cultures, and Nathan J. Regimbal and Laura Ortaliza for help with the preparation of the manuscript.

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