A simple fluorescence method for surface antigen phenotyping of lymphocytes undergoing DNA fragmentation

Journal of Immunological Methods, 154(1992)99-1()7

99

~'3 1992ElsevierSciencePublishersB.V. All rights reserved 01}22-1759/92/$05.01}

JIM 116422

A simple fluorescence method for surface antigen phenotyping

of lymphocytes undergoing DNA fragmentation Janet A. H a r d i n a, David H. Sherr '~, M a r y A n n D e M a r i a 6 a n d Peter A. Lopez ~,.6 "Departmentof Pathology. Harrard Medical School, and b Flow Cytometry Facility. Dana-FarberCancer Institute. Boston. MA. USA (Received5 March 1992,revisedreceived20 April 1992,accepted 21)April 1992)

Apoptosis, a metabolically active process of programmed cell death characterized by DNA fragmentation, is believed to play an important role in development of lymphocyte repertoires and in embryogenesis. Studies of this phenomenon would be greatly facilitated by the development of a simple assay capable of identifying and isolating intact apoptotic cells. A rapid fluorescence assay which identifies reIat:.vely small, intact cells containing fragmented DNA is described in this report. Thymocytes in which DNA fragmentation is induced by culture with or without dexamethasone are readily identified by their bright blue fluorescence after a 15 min treatment with Hoechst 33342, a DNA binding fluorochrome which diffuses through cell membranes. Since Hoechst 33342 staining does not require destruction of the cell membrane, it is possible to directly phenotype cell surface antigen expression on Hoechst 33342 bright lymphocytes by conventional immunofluorescence techniques and to evaluate membrane integrity of Hoechst 33342 bright cells by dye exclusion criteria. The advantages of this system are that it: (1) is rapid and simple, (2) quantitates the percentage of cells fragmenting their DNA and presumably undergoing apoptosis, (3) permits standard immunofluorescence staining of cell surface markers to identify even minor cell subsets of presumably apoptotic cells within heterogeneous populations, (4) provides the tools (fluorescence activated cell sorting) for purifying intact ceils containing fragmented DNA for further biochemical studies, and (5) provides a means for identifying cells which exclude vital dyes and in which DNA fragmentation will eventually result in cell death. Key words: Apoptosis;Thymocyte;Multicolorflow cytometry

Introduction

It is well accepted that cells die by at least two mechanisms, necrosis and apoptosis (Kerr et al., 1972). Necrosis is a passive process typified by cell dissolution and frequently initiated by

Correspondence to: D.H. Sherr, Departmentof Pathology, Harvard MedicalSchool,200 LongwoodAvenue,Boston, MA 02115, USA. Tel.: 617-432-1981.

metabolic poisons or by physical shock (Kerr, 1971; lshigami et al., 1992). In contrast, apoptosis is a metabolically active process associated with changes in cell and nuclear membrane morphology (Wylie et al., 1980; Shimizu et ai., 1990) and mediated by a Ca2+-dependent endonuclease which cleaves host DNA into oligonucleosomesized fragments (Wylie, 1980). Apoptosis, also referred to as programmed cell death, is believed to play a critical role in the development of T and B lymphocyte repertoires

(Liu et al., 1989; Smith et ai., 1989; Chang et al., 1991). Recognition of au~oantigens by developing lymphocytes results in the activation of the cell death program and the subsequent deletion of potentially patholr ~ic amoreactive clones. This process can be modeled in l'itro by cross-linking surface receptors on immature T or B:.cells with anti-CD3 or anti-lg antibodies respectively (Smith et ai., 1989; Benhamou, 1990). Whiie much has been learned about programmed cell death, studies in this critical area of lymphocyte development have been handicapped by the lack of a quantitative assay in which apoptotic cell subsets present in heterogenous populations can be phenotyped for cell surface antigen expression ~efore, during, and after delivery of the: cell death signal. Such an assay would make it possible to directly study programmed cell death in low nrnnbets of lymphocytes without prior enrichment of cell subsets (Chang et al., 1991). Herein is described a simple and rapid fluorescence method for quantitating the :m.,mber of mammalian cells in which DNA is cleaved into oligonucleosome-sized fragments, the imllmark of apoptosis. Hoechst (Ho) 33342, a DNA-binding benzimidazole dye shown to readily diffuse into intact, viable cells (Stohr and Vogt-Schaden, 1980; Crissman and Steinkamp, 1990; Latt and Langlois, 1990), was employed to identify thymocytes in which DNA fragmentation was effected by culture with or without dexamethasone (Cohen and Duke, 1984; Swat et ai., 1991; Telford et al., 1992). Since DNA staining with Ho 33342 was performed without disruption of the cell membrane, it was possible to phenotype cell surface antigen expression on cells containing fragmented DNA by conventional immunofluorescence techniques. Furthermore, by combining Ho 33342 with pro~idium iodide, a fluorochrome widely used to define the integrity of cell membranes, it was possible to identify cells which excluded propidium iodide but which contained fragmented DNA.

Materials

Mice C57BL/6 male mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Animals

were maintained according te the guidelines established by the Committee on Animals of the Harvard Medical Sehool and by those initiated by the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (DHHS Publication (NIH) 85-23, revised 1985).

Cell preparations Single cell suspensions of thymocytes were prepared by gently disrupting thymuses from 2 4-week-old donors with forceps. B cells were prepared by passing spleen cells from 4-6-week-old mice over nylon wool columns (Julius et al., 1973). Nylon wool adherent cells were then treated with a mixture of culture supernatants containing greater than 10/~g/ml monoclonal anti-Thy-l.2 antibodies (clones HO-13.4 and 30-H12, American Tissue Culture Collection, Rockville, MD) at room temperature for 30 min followed by incubation for 30 min at 37°C with 1/5 dilution of guinea pig complement (Peifreeze, Rogers, AK). Less than 3% of treated splenic populations were Thy-l.2 ÷. A slgM + B cell clone (JH1091), previously prepared in our laboratory, was also used as a bomogeneous source of B lymphoeytes. Induction of apoptosis Thymocytes were resuspended at a concentration of 4 × 106 celis/ml in Iscove's modified Dulbecco's medium (IMDM, Gibco/BRL,,Grand Island, NY) supplemented with 10% fetal calf serum (Hycione, Logan, UT), penicillin (50 U/ml; G i b c o / B R L ) , streptomycin (50 /Lg/ml; Gibco/BRL), L-glutamine (2 mM; Gibco/BRL) and gentamycin (50/~g/ml; Gibco/BRL). Cells were cultured in 25 cm 2 tissue culture flasks (Coming, Coming, NY) with or without dexamethasone (Sigma, St. Louis, MO) for 12 h at 37° in a humidified 10% CO 2 atmosphere. Cell viability was assessed by trypan blue and propidium iodide (PI, Sigma, 2.5 txg/ml) exclusion. Hoechst 33342 staining Fresh or cultured thymocytes were washed and adjusted to a concentration of 1.5 x 106 cells/ml in RPMI 1640 medium. In preliminary experiments the time of Ho 33342 (Molecular Probes, Eugene, OR) exposure (1-30 min), the tempera-

equiped with an argon ion laser (488 nm) for PE and PI excitation and an argon ion laser (360 nm) for Hoechst 33342 excitation. Ho 33342 fluorescence was detected through a 450 nm band pass filter (Omega Optical, Brattleboro, VT). PE or P1

ture during exposure (4°-37°C), and Ho 33342 concentration (0.25-2.0 p,g/ml) were varied. Incubation of cells for 15 min at 37°C with 1 / ~ g / m l Ho was found to be optimal for distinguishing Ho d"" from Ho bright populations. Cells were kept on ice after treatment to minimize further Ho :13342 uptake. Where indicated Ho 33342 stained cells were also treated for 10 min with 2 . 5 / z g / m l propidium iodide.

fluorescc,,c¢ was detected after being reflected 90° ~;ti'= a 560 short pass dichroic mirror and passage through a 575 nm band pass or 610 long pass filter, respectively. Forward angle light scatter from the 360 nm laser was detected through a 360 nm band pass filter. 90° light scatter from the 360 nm laser was detected after being reflected 90° with a 380 nm long pass filter. Fluorescence detectors were shielded from scattered laser light with 390 nm LP and 488 nm blocking filters.

lmmunofluorescence Cells were suspended at a concentration of 1.5 × 10 ~ cells/ml and incubated for 30 min on ice with RPMI containing 5% fetal calf serum and 5 / ~ g / m l PE-conjugated rat monoclonal antiThy-l.2 (Coulter, Hialeah, FL) or monoclonai isotype control (Southern Biotechnology, Birmingham, AL) antibody. Cells were washed two times in PBS containing 1% BSA and 0.02 M sodium azide and analyzed for Ho 33342 and PE-anti-Thy-l.2 antibody staining.

DNA fragmentation in agarose gels

Flow cytometry Flow cytometry and cell sorting were performed with a dual laser Coulter Epics 750 series flow cytometer (Coulter Electronics, Hialeah, FL)

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Thymocytes were washed three times in cold, 0.03 M Tris-buffered saline (pH 7.4). On the final wash 5 × l0 s or 2 × 10~ cells were pelleted in microfuge tubes and 20 p,I elution buffer containing 50 mM Tris-HCI, pH 8.0 (Sigma), 0.5% sodium lauryl sarkosinate (Sigma), 0.5 m g / m l proteinase K (Sigma), 10 mM EDTA (Sigma) were added. Cells were digested for 1 h at 50°C. 10/zl of 0.5 m g / m l RNAse ( G i b c o / B R L ) w e r e added and

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Fig. 1. Dose-dependentinduction of Hoechst 33342hrlgh~thymocyteswith dexamethasone.Thymocyteswere cultured for 12 h in medium with or without titered doses of dexamethasoneas indicated. Cells were harvested,tested for viabilityby trypan blue and PI exclusion, incubated for 15 rain at 37°C with 1 /.¢g/ml Hoechst 33342 and analyzed for Hoechst 33342 fluorescenceby flow cytometry (see Fig. 2). Lymphocyteswere gated into the analysesas dictated by forward and 90° light scatter parameters. Boxes indicate regionsdefiningHobrishl populations. A: fresh thymocytes;B: thymocytescultured for 12 h in media alone; C: thymocytes cultured for 12 h in media containing 10-'~ M dexamethasone;D: thymocytescultured for 12 h in media containing 10-s M dexamethasone;E: thymocytescultured for 12 h in media containing 10 - 7 M dexamethasone.

DNA extracts incubated for an additional h at 50°C. Extracts were heated to 70°C and 10 pA of warm loading mixture containing 10 mM E D T A (pH 8.0), 0.25% bromophenot blue ( G i b c o / B R L ) , 40% sucrose (Fisher, Fairlawn, NJ), and 1% agarose (FMC, Rockland, ME) was added. Extracts were loaded into dry wells of 10 cm, 4.5% Nusieve agarose (FMC) gels, allowed to harden. and electrophoreised at 70 V for 6 h in Trisa c e t a t e / E D T A running buffer (Sambrook et al.). 1 p.g of mspl digested pBR322 DNA (New England Biolabs, Beverly, MA) was run as a standard. Gels were stained by soaking in a 0.5 ~tg/ml ethidium bromide (Fisher) solution and were photographed under ultraviolet light.

Results Dexamethasone and related glucocorticoids have been shown to be potent inducers of apoptosis in thymocyte populations (Wylie, 1980; Cohen and Duke, 1984; Telford et al., 1992). To determine if cells containing fragmented D N A could be identified by Ho 33342 fluorescence patterns, thymocytes were cultured in medium

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alone or in medium containing 10 - 7 - 10 -') M dexamethasone. Cells were harvested after 12 h, treated with 1 ~.g/ml Ho 33342 at 37 ° for 15 min, and analyzed in a dual laser Coulter Epics 750 series flow cytometer. Ho 33342 fluorescence was plotted ag,.inst forward angle light scatter, a measure of cell size. Results from a representative experiment are presented in Fig. 1 and data from several experiments summarized in Table I. Ho 33342 stained all thymocytes to some extent. A minor subset of fresh thymoytes exhibit,~d a bright staining pattern (Fig. 1A). Ho bright thymocytes were smaller than Ho dull cells, as would be expected if at least some of these cells represented a population of apoptotic cells (Swat et al., 1991). The number of small, Ho bright thymocytes increased after culture for 12 h in medium alone (Fig. 1B), a result consistant with reports demonstrating that a minor population of thymocytes cultured in medium alone undergoes apoptosis (Sellins and Cohen, 1987; McConkey et ai., 1989; Swat et al., 1991). Addition of dexamethasone to thymocyte cultures resulted in a dose-dependent increase in small, Ho bright lymphocytes (Figs. IC, 1D and 1E). The percentage of viable cells in cultured populations, as assessed by try-

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Fig. 2. Fluorescenceactivated thymocyte sorting of Ho'jull, Ho~'ightand "Ho" ~ bright,subsets. Thymoctyeswere cultured for 12 h in medium with or without 10-~ M dexamethasone, harvested and stained with Hoeehst 33342. Thymocytes cultured with dexamethasone were sorted according to their level of Hoechst 33342 fluorescence. DNA from fresh thymocytes, thymocytes cultured without dexamethast~neand sorted thymocytesubsets was analyzed for DNA fragmentation by agarose gel electrophoresis (Fig. 3). A: fresh thymocyles; B: thymocytescultured for 12 h in media alone; C: thymocytescultured for 12 h in media containing 10"~ M dexamethasone with sorting gates overlayed; D: reanalysisof Hodull sorted cells; E: reanalysisof HO brighl sorted cells; F: Reanalysisof 'Hov~'~bright, ' sorted cells.

TABLE I DOSE-DEPENDENT INDUCTION OF HOECHST 33342h~ightTHYMOCYTES WITH DEXAMETHASONE ;' Thymocyte Irealment

Percent HObright thymocyles

Fresh thymocytes 12 h culture

9_4-2% (4) 35-+2%* (4)

Culture+ 10 -'~ M dexamethasone

40_+5% * (3)

Culture+ 10-s M dexamethasone Culture + 10-7 M dexamethasone

87_+6%* (2) 94 + 2% * (4)

I Thymocytes were cultured for 12 h in medium with or withoui titered doses of dexamethasone as indicated. Cells were harvested, tested for viability by trypan blue and PI exclusion, incubated for 15 rain al 37°C with 1/zg/ml Hoechst 33342 and analyzed for Hoechst 33342 fluorescence by flow cytometry (see Fig. I). Approximatc'y 85%, 83%, 75% and 60% of cells were viable in the media and IIV '~, l0 s l0 7 M dexamethasone-treated groups, respectively. Lymphocytes were gated into the analyses as dictated by forward and t~p light scatter parameters. An asterisk indicates a significant increase in Up hrlght thymocytes relative to fresh thymocytes, p < 0.(101.

p a n blue exclusion, was a p p r o x i m a t e l y 85%, 83%, 7 5 % a n d 6 0 % in t h e m e d i a a n d 10 -'j, l0 - s , a n d 10 - 7 M d e x a m e t h a s o n e - t r e a t e d c u l t u r e s respectively. T h e s e results are c o n s i s t e n t with t h e hyp o t h e s i s t h a t at least a p r o p o r t i o n of t h e H o bright l y m p h o c y t e s r e p r e s e n t apoptotic cells which, by dye exclusion criteria, are still viable. A p o p t o s i s is c h a r a c t e r i z e d by D N A f r a g m e n t a tion (Wylie, 1980; M c C o n k e y et al., 19911. T o directly correlate H o 33342 staining with D N A f r a g m e n t a t i o n , t h y m o c y t e s were c u l t u r e d with or w i t h o u t a low dose (10 -'j M) of d e x a m e t h a s o n e . A f t e r 12 h cells f r o m d e x a m e t h a s o n e t r e a t e d cult u r e s were s t a i n e d with H o 33342 a n d s o r t e d into HO dull, H o bright, a n d ' H o v~rybright, p o p u l a t i o n s (Fig. 2). D N A f r o m fresh t h y m o c y t e s a n d f r o m fluorcsc e n c e sorted s u b s e t s w a s extracted a n d elect r o p h o r e i s e d in a g a r o s e gels (Fig. 3). H o bright cells were s e g r e g a t e d into bright a n d ' v e r y bright' subp o p u l a t i o n s in this series o f e x p e r i m e n t s since s o m e h e t e r o g e n e i t y in t h e H o h~ight staining pattern was s u g g e s t e d in a p p r o x i m a t e l y 1 / 3 o f t h e experiments. C o n s i s t e n t with previous e x p e r i m e n t s , a significant increase in H o b~ight cells w a s o b s e r v e d following culture of t h y m o c y t e s in m e d i u m a l o n e or

in m e d i u m c o n t a i n i n g I() -'~ M d e x a m e t h a s o n e (Figs. 2 B a n d 2C). T h e high Icvel o f H o 33342 staining facilitated efficient .sorting of H o a~u, H o bright, a n d H o ~ry bright s u b p o p u l a t i o n s (Figs. 2 D , 2E, a n d 2 F ) . F r a g m e n t a t i o n of D N A from fresh thymocyte p o p u l a t i o n s c o n t a i n i n g 9 % H o hrigh~ cells was not d e t e c t e d in a g a r o s e gels (Fig. 3, lane l). However, a significant Icvel o f f r a g m e n t a t i o n was evident in 2 × I(P thymocytes c u l t u r e d in m e d i u m alone (38% HO bright:, lane 2) a n d , p e r h a p s to a g r e a t e r extent, in t h s m o c y t e s c u l t u r e d with 1 0 - " M dexa m e t h a s o n e (42% Hoh~ight; lane 3). Significantly, D N A from [-Io dun cells ~ppeared entirely intact (lane 4). In .sharp contrast, D N A from H o b~i~ht a n d H o ~'y hrighl cells h a d b e e n cleaved into

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Fig. 3. DNA fragmentation in Hoechst 33342 brlght thymi~zytes. DNA from cells sorted as described in Fig. 2 was extracted and electorphoreised in 4.5% Nusieve agarose gels. Sizes of DNA fragments in base pairs are listed in the left margin. Lane 1: DNA from 2 x 106 fresh thyml~.-'ytes (9% Hohtit~ht); lane 2: DNA from 2 x Ill ~' thymlcytes cultured without dexamethasone for 12 h (38% Hohrigh~); lane 3: DNA from

2x l0 t' thymocytes cultured with 10 '~ M dexamethasoilc for 12 h (42% HOhril~ht).~lane 4: DNA from 2× I[I~' dex~.iethasone-treated, tip dull thymocytes (0.4% Ho brit:hl); lane 5: DNA from 2x ll)~' dexamethasonc-treated, n o hrighl thymocytes (92% Hohright); lane 6: DNA fr~ml 5x His dcxamethasonctreated, ' ! lit "crybright,thymocytes (95% lip w~ bright).

104 oligonucleosome-sized DNA fragments (lanes 5 and 6). Given that DNA was extracted from only 5 × 10"s Ho very bright cells there were no obvious differences between the HO bright and Ho verybright subsets. These results demonstrate that most, if not all, of the thymccytes undergoing DNA fragmentation expressed the HO bright (and Ho ~er~bright) phenotype(s). Since Ho 35342 staining was performed with intact cells it seemed feasible to simultaneously phenotype cells for Ho 33342 staining and surface antigen expression. To simulate experimental conditions in which this protocol could be used to phenotype subsets of cells undergoing DNA fragmentation within heterogeneous populations, thymocytes were cultured for 12 h with 10-7 M dexamethasone. This dose induces DNA fragmentation in greater than 95% of thymocytes (Fig. IE). Cultured cells were then mixed in known percentages with T cell depicted splenic populations or with cloned B cells. Cell mixtures were stained with Ho 33342 followed by labelling with monoclonal PE-anti-Thy-l.2 or PE-isotype control antibodies and analyzed for Ho 33342 and PE fluorescence. Ho bright, Thy-l.2 + cells were readily detected in cultured thymocyte populations but not in nylon wool adherent, anti-Thy-l.2 + C treated splenic populations (Figs. 4A and 4C). (Thy-l.2- cells were offscale in the analyses presented in Fig. 4). Nearly 40% of the cell mixture consisting of 40% thymocytes and 60%

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Thy-l.2- spleen cells two color stained with Ho 33342 and PE-anti-Thy-l.2 antibody (Fig. 4B). Furthermore, a plot of the percent double positive cells versus the percent of thymocytes added to the mixture demonstrated that the percent Thy-l.2 +, Ho bdght cells detected was proportional to the percent dexamethasone-treated thymocytes added to the mixture (Figs. 5A and 5B). Therefore, this protocol is able to distinguish minor populations (i.e. as little as 1%) Ho bright (apoptotic) T cell subsets from HoUUlI,Thy-l.2- subsets in heterogeneous populations. One limitation of previously described DNA fragmentation assays is that they cannot distinguish DNA fragmented in response to specific programmed cell death signals from DNA fragmented as a result of nonspecific release of endonucleases during cell death. Unlike Ho 33342, PI is excluded from viable cells. This characteristic, plus the distinct emission wavelengths of PI and the ability to label intact cells containing fragmented DNA with Ho 33342, suggested the possibility of distinguishingapoptotic Ho bright, PIcells from Hobright: PI + e~!!S containing fragmented DNA. To test this hypothesis, thymocytes were cultured for 8-12 h in medium alone or in medium containing 10-~, 10 -8, or 10 -7 M dexamethasone. Cells were harvested and membrane permeability determined by trypan blue and PI exclusion. Aliquots of cells were also stained with Ho 33342 alone or Ho 33342 followed by propid-

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Fig. 4. Two color staining of heterogeneous populations with Hoechst 33342 and PE-anti-Thy-l.2 antibody. Thymocytes were cultured for 12 h in 10-7 M dcxamethasone, harvested and mixed in known concentrations with T cell-depleted splenic populations. Cell mixtures were two color stained with Hoechst 33342 and monoclonal PE-anti-Thy-].2 antibody or PE-isotype control antibody and analyzed for Hoechst 33342 and PE fluorescence. Lymphocytes were gated into the analyses as dictated by re.yard and 90° light scatter parameters. Boxes indicate regions defining |lo h'ight, Thy-l.2 + populations. Thy-L2- populations are

off, ale in these analyses.A: 100%dexamethasone-treatedthymocytes;B: 40% dexamethasone-treatedthymocytes+60%T cell depleted spleniccells:C: 100%T celldepletedspleniccells.

TABLE II TWO COLOR STAINING WITH HOECHST 33342 AND PROPIDIUM IODIDE " Thymocyte treatment

Twpan blue exclusion

PI stain on!y;

Ho stain only;

Ho 33342 and PI two color stain n o bright, PIHO hril~hl, PI ÷

H o dull,

8-12 h culture Culture+ 10-~)Dx Culture+ 10-s Dx

86+5 86±4 725:3 685: I

85+ 4 88+ 1 645:14 525:5

26±2 29+_3 84+_3 97+2

14+_3 14+_2 43+_7 52+_ I

75.+_2 71±2 17±3 3±2

C u l t u r e + 10 - 7 Dx

P! -

[-lo hrilthl

10±1 12±2 41+_9 44+ 1

PI-

a Thymocyteswere cuPmred for 8 or 12 h in medium with or without titered doses of dexametha~me (Dx) as indicated. Cells were harvested and viabilitydetermined by trypan blue exclusion. Aliquots were treated with Pi ('Pl stain only'), Hoechst 33M2 ('Ho stain only'), or Hoechst 33342 followed by P! ('Ho 33342 and PI two color stain'). Cells were analyzed for Hoechst 33342 and/or Pl fluorescence by flow cytometry. Data are pooled from two experiments.

ium iodide. Cell viability, as assessed by trypan blue exclusion, ranged from 68-86% and was dexamethasone dose-dependent (Table !!). The percentage of cells excluding PI closely paralleled that obtained with trypan blue. As previously shown, the proportion of n o bright cells detected by treatment with Ho 33342 alone was also dexamethasone dose-dependent and consistantly exceeded the percentage of dead cells. Two color staining with Ho 33342 and Pi revealed that approximately half of the n o bright cells excluded propidium iodide. Treatment of cells with Ho 33342 and P1 did not significantly affect the total percentage of either H O bright o r PI + cells. This result is consistent with reports that Ho 33342 and propidium iodide have distinct D N A binding sites (Crissman and Steinkamp, 1990; Latt and Langlois, 1990) and indicates that the lack of PI staining of n o bright populations was not due to Ho 33342 competition for propidium iodide D N A binding sites. The results demonstrate the ability of this system to identify intact P I - cells destined for cell death by apoptosis.

Discussion Apoptosis is ar. important physiologic mechanism involved in the development of lymphocyte repertoires (Smith et al., 1989; Benhamou et al., 1990; Chang et al., 1991; McConkey et al., 1991) and in embryogenesis (Kerr et al., 1972). Numerous studies have attempted to evaluate cellular targets of and mechanisms responsible for this

physiologically relevant form of programmed cell death. These studies have been slowed by the lack of a rapid and simple method for identifying and isolating intact apoptotic cells. Herein we describe a simple, fluorescence method which appears to bridge this technologic gap. Several results supported the hypothesis that the DNA-specific dye, Hoechst 33342, detects cells undergoing apoptosis. First, the number of thymocytes staining brightly with Hocchst 33342 increased rapidly after culture and in proportion to the level of exposure to dexamethasone, a known inducer of apoptosis in thymocytes (Cohen and Duke, 1984). Secondly, as would be expected of apoptotic cells, dexamethasone-treated thymocytes, a significant fraction of which excluded trypan blue and PI, were smaller thorn untreated populations. Most importantly, cultured dexamethasone-treated thymocytes, .sorted on the basis of their bright Hoechst 33342 staining pattern, contained all of the fragmented DNA detectable in dexamethasone-treated populations as visualized by agarose gel electrophoreisis. This system builds on the studies of other investigators in which intact apoptotie cells were defined in part by forward and 90° light scatter characteristics (Swat et al., 1991). As would be expected from previous studies Ho bright (apoptotic) cells were relatively small (Figures 1 and 2). Therefore, changes in both cell morphology (forward and 90 ° light scatter characteristics) and chromatin structure (Hoechst 33342 staining profiles) can be applied simultaneously to better define apoptotic cells. Another advantage of this

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lymphocyte mixtures is proportional to the percentage of dexamethsone treated thymocytes added. Thymocytes were cultured for 12 h in 10 -7 M dexamethasone, harvested and mixed in known concentrations with T cell-depleted splenic populations (experiment I) or with cloned B cells (experiments 2 and 3). Cell mixtures were two color stained with Hoechst 33342 and PE-anti-Thy-l.2 antibody and analyzed for Hoechst 33342 and PE fluorescence (see Fig. 4).

system is that it can identify minor subsets of apoptotic cells within populations containing cells of various sizes and morphology. While this ability is not of particular advantage when studying relatively homogeneous populations such as thymocytes, it would be of particular value when assessing apoptosis in more heterogenous organs such as bone marrow and spleen.

The mechanism by which Hoechst 33342, a benzimidazole dye, preferentially stains cells undergoing DNA fragmentation is unknown. It is known that, unlike other DNA-binding dyes such as propidium iodide, actinomycin D, and acridine orange which intercalate into DNA, Hoechst 33342 binds to external DNA domains in minor grooves (Crissman and Steinkamp, 1990; Latt and Langlois, 1990). It is possible that DNA fragmentation exposes more of the A-T base pairs to which benzimidazole dyes preferentially bind (Mueller and Gautier, 1975) resulting in a brighter stain than that observed in unaffected Ho °u" cells. Supporting this hypothesis is the observation that the intensity of benzimidazole staining correlates with chromatin structure (Stokke and Steen, 1986). Furthermore, Hoechst 33342 readily enters viable cells (Crissman and Steinkamp, 1990) whereas propidium iodide, a highly polar dye, is impermeable to viable cells (Latt and Langlois, 1990). Therefore, both the distinct DNA binding characteristics and the characteristic cell diffusion properties of Hoechst 33342 may account for the staining patterns observed in this study. Indeed, we have taken advantage of the differences between Hoechst 33342 and propidium iodide to two color stain dexamethasone-treated thymocytes with Hoechst 33342 and PI in a fashion analagous to that originally described by Stohr and Vogt-Schaden 11980). These experiments demonstrated that approximately half of the Ho bright cells excluded PI. These results portend the ability to distinguish apoptotic ceils, in which DNA fragmentation results in cell death, from dead cells in which DNA fragmenh:tion is a consequence of cell death. This system offers some important advantages over previously described technologies. First, unlike gel electrophoresis in which DNA fragmentation is difficult to quantitate, Hoechst 33342 staining quantitates the percent Ho bright and presumably apoptotic cells in heterogeneous cell populations. Secondly, minor populations of Ho bright cells can he readily detected by flow cytomctry. Even assays which quantitate DNA fragmentation in heterogeneous cell populations (Sellins and Cohen, 1987; Kizaki et al., 1989; McConkey et al., 1991) cannot accurately determine what percentage of cells have been affected.

Third, as stated above, dead cells containing fragm e n t e d D N A can be gated out of analyses or cell sorts, thereby eliminating a potentially misleading artifact. Fourth, and p e r h a p s most significantly, this system allows immunoflaorescence phenotyping of cell surface antigens on H o br~ghl cell subsets without prior cell purification steps which in and of themselves could affect apoptosis. In eddition to facilitating the characterization of small numbers of presumably apoptotic cells, this system will enable us to purify intact cell subsets by fluorescence activated cell sorting for f u r t h e r biochemical analyses.

Acknowledgements This work was s u p p o r t e d by grants from the Elsa U. Pardee F o u n d a t i o n and from the National Institute of Allergy and Infectious Disease ( A I 23978) and by a Feasibility Study from the Massachusetts Institute of Technology, C e n t e r for Environmental Health Sciences. J.A.H. is a fellow of T h e Leukemia R e s e a r c h Foundation.

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