Dactinomycin – Akti 1 2

Multiparametric flow cytometric analysis of biochemical and functional events associated with apoptosis and oncosis using the 7-aminoactinomycin D assay
Herve´ Lecoeur, Luzia Maria de Oliveira-Pinto, Marie-Lise Gougeon*
De´partement de Me´decine Mole´culaire, Institut Pasteur, 28 Rue du Dr. Roux, 75724 Paris Cedex 15, France

Abstract

Apoptosis and primary necrosis are the two modes of cell death induced by a lethal injury. The majority of structural and biochemical events occurring during cell death can be analysed by flow cytometry. The 7-aminoactinomycin D (7-AAD) assay can be used to detect the loss of membrane integrity during apoptosis of murine thymocytes and human peripheral lymphocytes. We describe here new applications of the 7-AAD assay. It can be applied to a variety of cell lines of different origins, including adherent cell lines, and it allows the co-detection of lipidic antigens such as phosphatidylserine (PS) residues, and biochemical processes linked to apoptosis, such as the loss of mitochondrial transmembrane potential, cardiolipin peroxidation, the expression of the 7A6 mitochondrial antigen and DNA fragmentation. Thus, this assay is a noninvasive method particularly adapted to the analysis of biochemical events associated with cell death. Finally, we show that this assay is not specific for apoptosis since it detects oncosis, the early stage of primary necrosis. D 2002 Elsevier Science B.V. All rights reserved.

Keywords: 7-AAD; Flow cytometry; Apoptosis; Oncosis

1. Introduction

Apoptosis is an active mechanism of cell suicide that takes part in many immunological events includ-

Abbreviations: 7-AAD, 7-aminoactinomycin D; DiOC6(3), 3,3V-dihexyloxacarbocyanine iodide; FSC, forward scatter; HIV, human immunodeficiency virus; MFI, mean of fluorescence in- tensity; NAO, 10-N-nonyl-3,6-bis (dimethylamino) acridine; PBMCs, peripheral blood mononuclear cells; PFA, paraformalde- hyde; PI, propidium iodide; PMA, phorbol 12-myristate acetate; PS, phosphatidylserine; SSC, side scatter.
* Corresponding author. Tel.: +33-1-45-68-89-07; fax: +33-1- 45-68-89-09.
E-mail address: [email protected] (M.-L. Gougeon).

ing intrathymic selection of the T cell repertoire, CTL- induced cytotoxicity or homeostatic regulation after antigen stimulation (Arends and Wyllie, 1991; Kagi et al., 1994; Lenardo et al., 1999). This type of cell death is associated with profound structural changes and biochemical events leading to irreversible cell destruc- tion. Morphological alterations include cell shrinkage because of disordered volume regulation (Maeno et al., 2000), loss of membrane integrity (Ormerod et al., 1993), alteration in mitochondrial structure, chromatin condensation and emission of apoptotic bodies (Dar- zynkiewicz et al., 1992). Functional alterations and biochemical events include the loss of mitochondrial transmembrane potential (Dwm) (Petit et al., 1995;

Kroemer et al., 1997), loss of lipid asymmetry man- ifested by the translocation of phosphatidylserine (PS) residues to the outer layer of the plasma membrane (Koopman et al., 1994), cardiolipin peroxidation, activation of caspases (Zheng and Flavell, 2000) and DNA fragmentation into oligonucleosomal fragments (Walker et al., 1995). Primary necrosis, or accidental cell death (Darzynkiewicz et al., 1997), is a passive, catabolic and degenerative process that generally represents a response to gross injury and can be in- duced either by an overdose of cytotoxic agents, by serious injury or intracellular pathogens (Oshimi et al., 1996; Leist et al., 1997; Darzynkiewicz et al., 1997; Palomba et al., 1999). Majno and Joris (1995) have pointed out that cells committed to primary necrosis pass through a pre-lethal process, called oncosis, dur- ing which cells swell but preserve their plasma mem- brane integrity.
The majority of structural and biochemical events occurring during cell death can be analysed by flow cytometry. Changes in cell volume and granularity are easily detected with the FSC/SSC parameters. Increased membrane permeability can be evidenced by the incorporation of fluorescent intercalative DNA dyes such as Hoescht 33342 (Ormerod et al., 1993), ethidium bromide (EB) (Olivier, 1995), propidium io- dide (PI) (Ormerod et al., 1993; Zamai et al., 2001), YOPRO-1 (Idziorek et al., 1995) or 7-aminoactino- mycin D (7-AAD) (Schmid et al., 1992). 7-AAD is an actinomycin D (AD) analog able to bind to DNA, which was initially used in chromosome analysis (Modest and Sengupta, 1974), cell cycle studies (Zelenin et al., 1984; Rabinovitch et al., 1986) and further used to quantify apoptosis (Schmid et al., 1992). In addition, to be readily quantifying apoptosis, 7-AAD staining offers the advantage of discriminating between early and late apoptotic cells and it can be combined with other staining procedures to study apoptosis in a multiparametric approach at the single cell level (Schmid et al., 1994a,b; Lecoeur et al., 1997, 1998). In the present study, we show new data on multiparametric applications of the 7-AAD assay in the co-detection of molecules or events associated with apoptosis, such as PS expression on the plasma membrane, intracellular expression of mitochondrial 7A6 protein, loss of Dwm, activation of caspases or DNA fragmentation. In addition, we describe how 7- AAD staining can be used to quantify apoptosis in

adherent cells and to analyse oncosis, the early phase of primary necrosis.

2. Materials and methods

2.1. Culture and induction of apoptosis in peripheral blood lymphocytes and cell lines

Jurkat T cell line and U937 cells were cultured at
5 105 cells/ml at 37 jC in a 5% CO2 humidified atmosphere in RPMI medium (Gibco, Paisley, UK) supplemented with 10% heat-inactivated fetal calf serum (Institut Jacques Boy, Reims, France), 10 IU/ ml penicillin, 10 Ag/ml streptomycin, 10 mM HEPES and 1 mM L-glutamine (Sigma, St. Louis, MO) (complete medium). Peripheral blood mononuclear cells (PBMCs) from healthy human volunteers or HIV + patients were isolated from heparinized blood samples after centrifugation on a Ficoll-Hypaque density gradient (Pharmacia, Uppsala, Sweden). PBMCs were washed and cultured overnight in com- plete medium at 1 106 cells/ml. HeLa cells were cultured in complete DMEM (Gibco) in 75-ml flasks and submitted to stimuli at 75% confluence. The murine MMHD3 cell line was cultured in DMEM/ Ham’s F12 medium supplemented with 10% FCS, 50 ng/ml EGF and a mixture of insulin, transferrin and selenium (Sigma), as previously described (Terradillos et al., 1998).
Apoptosis in PBMCs was induced either by over- night stimulation with phorbol 12-myristate acetate (PMA, 50 ng/ml, Sigma) plus ionomycin (Iono, 250 ng/ml, Sigma), as previously reported (Ledru et al., 1998), or by coated anti-CD3 mAbs (IoT-3, Immuno- tech, Marseille, France) plus 0.05 Ag/ml interleukin 2 (IL-2, R&D Systems, Abingdon, UK), or by coated anti-CD95 mAbs (CH-11, Immunotech). In PMA– Iono-treated samples, 10 Ag/ml Brefeldin A (Sigma) was added in the culture to further detect intracellular cytokine synthesis, as reported (Ledru et al., 1998). Apoptosis in U937 cells was induced by 7-day star- vation and in CEM cells and HeLa cells by 2 Ag/ml cycloheximide (CHX, Sigma). Apoptosis in MMHD3 cells was induced after transfection of human FADD cDNA vector (pcDNA3-AU-1-FADD) (Terradillos et al., 1998). Primary necrosis was induced after a 24-h treatment with 1% sodium azide (NaN3, Sigma),

hypotonic shock with distilled water or after 3 min of hyperthermia at 56 jC, as previously described (Lecoeur et al., 2001a,b).

2.2. The 7-aminoactinomycin D (7-AAD) assay: concomitant detection of surface and intracellular molecules

7-AAD (Sigma) is a fluorescent DNA dye that selectively binds to GC regions of the DNA (Modest and Sengupta, 1974). During the cell death process, the plasma membrane is progressively altered and becomes permeable to the dye. At high concentrations of 7-AAD (20 Ag/ml), both early (7-AADLo) and late (7-AADHi) apoptotic cells can be detected with the 7- AAD assay (Schmid et al., 1994a). The 7-AAD assay has been initially described for the detection and quantification of apoptosis (Schmid et al., 1992; Lecoeur and Gougeon, 1996). Briefly, cells were incubated in PBS containing 20 Ag/ml of 7-AAD for 20 min at + 4 jC in the dark. Then samples were washed and resuspended in PBS– 1% BSA– 0.1% NaN3 containing 20 Ag/ml of nonfluorescent actino- mycin D (AD, Sigma) to avoid the progressive release of 7-AAD (Lecoeur et al., 1997). Finally, cells were fixed in AD buffer containing 1% paraformaldehyde (PFA) for 20 min at + 4 jC, and samples were immediately analysed. 7-AAD emission was detected in the FL-3 channel (>650 nm).
The 7-AAD assay was further adapted to simulta- neous labeling of both surface (Schmid et al., 1994b; Lecoeur et al., 1997) and intracellular antigens (Lecoeur et al., 1998, 2001b). Extracellular staining procedures were performed during the 7-AAD stain- ing step with anti-CD45RO (IgG2a, UCHL-1, clone P3/NS-1/1-Ag4-1) and anti-CD8 (IgG1k clone SK1) mAbs, both purchased from Becton Dickinson, San Jose, CA. Intracellular staining procedures were per- formed after PFA fixation, and cells were incubated with monoclonal or polyclonal antibodies specific for the active form of caspase-3 (PharMingen, Le Pont de Claix, France), the 7A6 protein (clone Apo2.7, Immu- notech) or IFNg (IgG2b, clone 25-723.11, Becton Dickinson), diluted 1/20 in 0.05% Saponin Quillaja bark (Sigma) in PBS– 1% BSA– 0.1% NaN3 contain- ing 20 Ag/ml AD. After a 30-min incubation, cell samples were washed and fixed in PBS– 1% PFA– NaN3– AD buffer (Lecoeur et al., 1998, 2001b). The

active form of caspase-8 was detected by fixation of the specific carboxyfluorescein-labeled caspase inhib- itor FAM-LETD-FMK (CaspaTagk Caspase-8 Activ- ity Kit, Intergen, Oxford, UK). Briefly, after 7-AAD staining, cells were submitted to FAM-LETD-FMK staining for 1 h at 37 jC in a 5% CO2 humidified atmosphere, washed and fixed in a specific fixative buffer (CaspaTagk Caspase-8 Activity Kit).
Flow cytometry analyses were performed on a FACSCalibur cytometer (Becton Dickinson), equip- ped with a single 488-nm argon laser. For each sample, 10,000 events were recorded in list mode and regis- tered on logarithmic scales. Analysis was performed with the Cell Questk software (BDIS).

2.3. Detection of mitochondrial alterations

A drop in the mitochondrial transmembrane poten- tial Dwm was analysed on unfixed cells using 3,3V- dihexyloxacarbocyanine iodide ((DiOC6(3), Molecu- lar Probes, Eugene, OR), a lipophilic dye sequestered into the mitochondrial matrix according to the Nernst equation (Petit et al., 1995). A total of 5 105 cells were stained with 0.1 AM DiOC6(3) and 7-AAD in 100 Al PBS for 30 min at 37 jC, and were immedi- ately acquired in their staining solution on a FACS- Calibur (BD). DiOC6(3) fluorescence was collected on the FL-1 channel (bandpass, 530 nm; bandwidth, 30 nm) as previously described (Petit et al., 1995).
Alterations of mitochondrial structure (cardiolipin peroxidation) were evaluated following 10-N-nonyl- 3,6-bis (dimethylamino) acridine (NAO, Molecular Probes) incorporation into unfixed cell samples. Af- ter 7-AAD staining, 5 105 cells were stained with 4 AM NAO in 100 Al PBS for 30 min at 37 jC, and were immediately acquired in their staining solution on a FACSCalibur (BD) (Petit et al., 1995). NAO fluorescence was collected on the FL-2 channel (bandpass, 585 nm; bandwidth, 42 nm).

2.4. Detection of DNA fragmentation by the TUNEL assay

DNA fragmentation occurring during cell death was detected by the sensitive terminal deoxynucleo- tidyl transferase dUTP nick end labeling (TUNEL) assay (Gorczyca et al., 1993). For 15 min, 5 105 cells were fixed in 1% PFA, washed in PBS contain-

ing 20 Ag/ml AD, permeabilized in 70% ice-cold ethanol + 20 Ag/ml AD for 5 min and submitted to the nick end labeling in a mixture from the ApoDirect Kit (PharMingen). The detection of 3VOH end was performed by FITC-conjugated dUTP.

2.5. Detection of the loss of membrane asymmetry by Annexin V staining

During apoptosis, the plasma membrane loses its asymmetry and phosphatidylserine (PS) residues present in the inner leaflet are translocated to the outer leaflet (Vermes et al., 1995). Cells were initially stained with 7-AAD and further stained by FITC- Annexin V in a Ca2+ -enriched binding buffer (Apop- tosis detection kit, R&D Systems) for 15 min at 20 jC (Vermes et al., 1995). In some experiments, PI was added to Annexin V-stained samples according to manufacturer’s recommendations. FITC-conjugated Annexin V and PI emissions were detected in the FL-1 and FL-2 channels, respectively.

2.6. Optical and electron microscopy

Ultrastructural studies were performed by trans- mission electron microscopy. A total of 1 106 cells were submitted to fixation with 2.5% glutaraldehyde in Soe¨rensen buffer (phosphate, 0.1 M, pH = 7.4), and further dehydrated in a series of ethanol solutions (30 – 100%). Finally, cells were embedded in epoxy resin. Sections were performed with a Reichter– Jung ultramicrotome before examination. Cells were exam- ined with a Jeol JEM 1200 EX electron microscope at
× 6000 magnification.

3. Results

3.1. Apoptosis quantification in human peripheral T lymphocytes and T cell lines

Most of the cellular and molecular events associ- ated with apoptosis, such as structural and functional alterations in mitochondria, exposure of PS on the outer layer of plasma membrane, increased membrane permeability, activation of caspases, nucleus and DNA fragmentation and, finally, blebbing of apoptotic bodies, can be analysed by flow cytometry. The loss

of plasma membrane integrity can be evidenced by cell permeability to vital fluorescent DNA dyes such as YOPRO-1 (Idziorek et al., 1995) or 7-AAD (Schmid et al., 1992). 7-AAD staining permits the detection of apoptotic cells in a variety of lymphoid populations such as murine thymocytes, peripheral blood lymphocytes from humans and nonhuman pri- mates (Schmid et al., 1992; Lecoeur and Gougeon, 1996; Gougeon et al., 1997) or cell lines. Fig. 1A shows representative stainings of PBMC from HIV- infected persons, previously shown to be primed for spontaneous apoptosis induced by overnight culture in medium (Gougeon et al., 1996). 7-AAD can also be used to detect apoptotic cells in T cell lines, i.e. CEM clone 13 and Jurkat cells (Fig. 1A) incubated in the presence of cycloheximide (CHX) which blocks syn- thesis of short-lived protective proteins (Lemaire et al., 1999). 7-AAD staining facilitates discrimination between early and late apoptotic cells when used at high concentration (20 Ag/ml) (Schmid et al., 1994a), and both subsets can be identified in a 7-AAD– MFI/ FSC dot plot, with early apoptotic cells showing lower MFI and higher FSC than late apoptotic cells (Fig. 1A). Note that 7-AAD/FSC dot plots show different patterns of dispersion of early and late apoptotic cells depending on the type of cells tested. A comparison of the 7-AAD assay with the Annexin V/PI assay (Vermes et al., 1995) is shown in Fig. 1B for Jurkat cells incubated with CHX. The percentage of early apoptotic cells, defined by low 7-AAD staining, is
similar to the percentage of Annexin V + PI — , where-
as late apoptotic cells incorporate high amount of
7-AAD and are permeable to PI. More importantly, an excellent correlation was obtained between both tests for early and late apoptotic cells (Fig. 1C). These data suggest that early PS exposure is already associated with a moderate but significant loss of membrane in- tegrity, and they confirm previous observations indi- cating that, like the Annexin V/PI assay, the 7-AAD assay is appropriate for the detection of early stages of apoptosis (Lecoeur et al., 1997).

3.2. Apoptosis quantification in adherent cells

Initially, the 7-AAD assay was described to meas- ure apoptosis on lymphoid cells in suspension (Schmid et al., 1992), whereas the TUNEL assay and the Annexin V/PI assay were used to measure

Fig. 1. Apoptosis quantification by both 7-AAD and Annexin V/PI assays. (A) Patterns of 7-AAD staining in PBMCs from an AIDS patient cultured overnight in medium, Jurkat cells treated for 16 h with 2 Ag/ml CHX or U937 cells after medium starvation. Living, early and late apoptotic cells were defined according to the level of 7-AAD staining. The mean of fluorescence intensity (MFI) of 7-AAD staining and mean FSC are indicated for each subset. (B) Representative FSC/7-AAD and Annexin V/PI dot plots of Jurkat cells cultured for 16 h either in medium or with 2 Ag/ml CHX. (C) Correlations between the percentage of early or late apoptotic cells quantified either by the 7-AAD assay or the Annexin V/PI assay on 26 samples of Jurkat cells. Values of p and r2 are indicated.

apoptosis on adherent cells (Grasl-Kraup et al., 1995; Van Engeland et al., 1996). To determine whether 7- AAD staining was appropriate for the detection of apoptosis in adherent cell populations, we set up a procedure, described in Fig. 2B, which includes apoptotic cells that progressively detach from the adherent cell layer. Indeed, Fig. 2A shows that, following a 6-h incubation of HeLa cells with CHX, the supernatant contains a majority of apoptotic cells (7-AAD + ), whereas attached cells mostly include living cells. To quantitate accurately the total number of apoptotic cells in a culture of adherent cells, supernatant was collected first, adherent cells were washed and both cell preparations were stained with

7-AAD. Adherent cells were then detached from the flask with EDTA (trypsin was avoided because it was shown to alter surface molecule expression, prevent- ing further phenotypic analysis of apoptotic cells, Corver et al., 1995) and after washes, both cell pop- ulations were mixed and analysed on a flow cytometer (Fig. 2B). This procedure has been applied on the murine hepatocyte cell line MMHD3 FADD, trans- fected with an expression vector for FADD, and on the cell line MMHD3 pcDNA3, transfected with the empty vector pcDNA3 (Terradillos et al., 1998). 7- AAD/FSC dot plots are shown in Fig. 2C. Apoptotic cells could be identified by their ability to incorporate 7-AAD, and a parallel analysis performed with

Fig. 2. Detection of apoptosis in adherent cells by the 7-AAD assay. (A) Quantification of apoptotic cells in the supernatant and adherent fractions of HeLa cells cultured for 6 h in the presence of 2 Ag/ml CHX. (B) Procedure for apoptosis quantification in adherent cell lines with the 7-AAD assay. (C) FSC/7-AAD dot plots of adherent cell lines: hepatic MMHD3 cells were transfected with the expression vector for FADD or the empty vector pcDNA3, cultured for 48 h in medium and then submitted to the staining procedure described in part B. A similar procedure was applied to HeLa cells treated with 2 Ag/ml CHX for 12 h.

Hoescht 33342 gave similar data (Pollicino et al., 1998). However, 7-AAD staining could not discrim- inate between early and late apoptotic cells. Similar observations were made with the cervical carcinoma HeLa cell line submitted to CHX (Fig. 2C).

3.3. Phenotypic characterization of apoptotic cells

In a complex population such as PBMC, it is essential to be able to phenotype cell subsets involved in the cell death process. Indeed, it avoids cell sorting before the induction of apoptosis that could introduce pitfalls since purified cells may behave differently from the total population because of the possible survival/ apoptotic role of some cell subsets, such as cytokine-

producing cells or antigen-presenting cells. We have shown previously (Lecoeur et al., 1997, 1998) that the 7-AAD assay preserves the structure of the plasma membrane and the expression of surface antigens, in contrast to other assays which require drastic mem- brane permeabilization (Jonker, 1993; Douglas et al., 1995). Fig. 3A and B shows that the activation markers CD45RO and HLA-DR can be co-detected on apop- totic lymphocytes stained with 7-AAD. It is notewor- thy that apoptotic cells are more weakly stained with specific mAbs compared to living cells, and this decreased antibody-binding capacity is detected on early apoptotic cells (7-AADLo) and more pronounced on late apoptotic cells (Fig. 3B). We previously re- ported similar observations for CD4, CD8 or CD19

Fig. 3. Phenotypic characterization of apoptotic cells. (A) Detection of apoptosis within CD45RO expressing cells in PBMCs from an AIDS patient cultured overnight in medium. Early and late apoptotic cells are indicated by grey and black arrows, respectively. Note the decrease in CD45R0 MFI on apoptotic cells. An FSC-CD45RO staining dot plot is also shown. (B) Decrease in the antibody-binding capacity (ABC) of apoptotic cells related to cell shrinkage (FSC). MFI of CD8, CD45RO and HLA-DR surface molecules in living, early and late apoptotic cells is plotted against the mean FSC. (C) PBMCs from an AIDS patient were cultured overnight in medium, and external exposure of PS on the outer layer of plasma membrane was analysed on apoptotic cells by combining Annexin V/7-AAD staining. (D) MMHD3 transfected with an expression vector coding for both the pro-apoptotic HBx protein and the murine H2Kd molecule was tested for apoptosis following 48-h
incubation in medium. Transfected cells were targeted by PE-conjugated anti-H2Kd mAbs, and apoptosis was quantified in this subset. The percentage of cells in each quadrant is indicated. Apoptosis in H2Kd + and H2Kd — was calculated and also indicated.

molecules (Lecoeur et al., 1997). Fig. 3A also shows that phenotyping of apoptotic cells can be performed by analysing surface molecule expression on cells with a decreased FSC since apoptotic cells undergo early shrinkage (Maeno et al., 2000). Fig. 3C shows that the 7-AAD assay permits the phenotyping of trans- fected cells. The adherent cells MMHD3 were trans- fected with an expression vector for both the pro- apoptotic HBx protein and the murine H2Kd molecule, and consequently, HBx-transfected cells expressed H2Kd molecules on their surface and these could be detected with specific mAbs. 7-AAD staining of these cells following a short-term culture showed that H2Kd + -transfected cells were more sensitive to apop- tosis (16.3%) compared to nontransfected cells (6.7%), confirming that HBx is an inducer of apoptosis (Terra- dillos et al., 1998; Pollicino et al., 1998).
Finally, we asked whether 7-AAD staining allows the co-detection of surface phospholipids, particularly phosphatidylserine (PS) residues. PBMCs from an AIDS patient were cultured overnight in medium, and external exposure of PS on the outer layer of plasma membrane was analysed on apoptotic cells. As shown in Fig. 3D, co-staining with 7-AAD and An- nexin V shows that early apoptotic cells (7-AADLo cells) exhibit significant PS exposure (MFI Annexin V = 2734) in contrast to living cells (7-AADneg cells MFI = 53). PS externalization is more important at the end of the apoptotic process (7-AADHi cells, MFI Annexin V = 4167). These results clearly demonstrate that Annexin V + cells have already lost their mem- brane integrity, confirming data from Fig. 1B.

3.4. Detection of alterations in mitochondria, activa- tion of caspases and DNA fragmentation in apoptotic cells

Apoptosis involves numerous biochemical events, including peroxidation of cardiolipin, dissipation of mitochondrial transmembrane potential (Dwm), expression of mitochondrial 7A6 protein, activation of caspases and DNA fragmentation (Walker et al., 1995; Petit et al., 1995; Kroemer et al., 1997; Zheng and Flavell, 2000). These events can be detected by flow cytometry using nonyl acridine orange (NAO), the DiOC6(3) cationic molecule, APO2.7 mAb, anti- caspase-3 polyclonal antibodies, or FITC-conjugated dUTP (TUNEL assay).

Fig. 4 shows representative results. Peroxidation of cardiolipin was tested on unfixed samples of Jurkat cells induced to apoptosis by a 5-h incubation with anti-CD95 mAbs and stained with 7-AAD and NAO (Fig. 4A). In contrast to living cells, which incorpo- rate high levels of NAO (MFI = 738), both early and late apoptotic cells exhibit lower staining with NAO as a consequence of significant cardiolipin peroxida- tion. Fig. 4B shows that the shift from living to early apoptotic cells is accompanied by a drop in Dwm, as indicated by the decreased ability of mitochondria to accumulate the cationic dye DiOC6(3), and this alter- ation is more pronounced in late apoptotic cells. Mitochondrial changes may also be documented by the expression of 7A6 protein, specifically expressed in mitochondria during apoptosis (Zhang et al., 1996). Detection of intracellular molecules within apoptotic cells can be achieved provided cell permeabilization is smooth. Indeed, by comparing various detergents, we found that Saponin Quillaja Bark, a steroid-based class B detergent that creates pore-like structures in cell membranes (Bomford et al., 1992), is the more appropriate (Lecoeur et al., 1998). Fig. 4C shows representative staining of intracellular 7A6 detected with APO2.7 mAb combined with 7-AAD on per- meabilized CEM 13 cells treated with 10 Ag/ml actinomycin D. 7A6 protein is expressed in 7-AADLo cells and to a similar extent in late apoptotic cells. 7A6 is also detected in Jurkat T cells killed in the context of CD95-dependent cell-mediated cytotoxicity (Lecoeur et al., 2001b).
Apoptosis involves the activation of a cascade of caspases (Cohen, 1999; Los et al., 1999). The active form of initiator caspase-8 can be detected by flow cytometry using FAM-LETD-FMK. This inhibitor irreversibly binds to active caspase-8 but not to the pro-enzyme. Because of its conjugation to FAM, cells expressing intracellular-activated caspase-8 express green fluorescence. Fig. 4D shows the co- expression of caspase-8 and 7-AAD on PBMCs from a control donor following 16 h of stimulation with anti-CD3 mAbs plus IL-2. Interestingly, in addition to being expressed on apoptotic cells (7-AADHi or 7-AADLo), caspase-8 was detected in a fraction of living cells. Caspase-3 is an effector caspase in- volved in the response to numerous stimuli (Cohen, 1999; Los et al., 1999; Zheng and Flavell, 2000). The active form of this enzyme can be detected by

Fig. 4. Detection of biochemical events associated to apoptosis. (A– C) Co-detection of apoptosis and mitochondrial alterations in Jurkat cells treated with anti-CD95 mAbs for 5 h. 7-AAD staining was combined to the detection of cardiolipin peroxidation by NAO incorporation (part A), the detection of the loss of mitochondrial transmembrane potential by DiOC6(3) staining (part B) and the expression of mitochondrial 7A6 protein by PE-conjugated APO2.7 mAbs (part C). (D– E) Co-detection of apoptosis and caspase activation. Active caspase-8 expression was detected in human PBMCs from a control donor following 16-h activation with anti-CD3 mAbs. Active caspase-3 expression was detected in human PBMCs from an HIV-infected subject treated under the same conditions. Grey and black arrows indicate early and late apoptotic cells, respectively. (F– G) Co-detection of apoptosis and DNA alterations in lymphocytes from a control donor, freshly isolated (ex vivo) or submitted to PMA– ionomycin stimulation for 16 h. DNA condensation/fragmentation was assessed by AO incorporation (F). Histograms represent AO incorporation of gated 7-AADneg (grey line), 7-AADLo (dotted line) and 7-AADHi cells (bold line). (G) DNA fragmentation was assessed by the TUNEL assay. Mean MFI of dUTP incorporation in three independent experiments is plotted against mean MFI of 7-AAD staining.

flow cytometry with specific polyclonal antibodies, as is shown in Fig. 4E, using Jurkat cells treated for
5 h with anti-CD95 mAbs. Active caspase-3 was

detected within 7-AADLo cells, suggesting that this enzyme is also involved in the early steps of cell death.

Chromatin condensation and DNA fragmentation are classical features of apoptotic cells in a great variety of cell types (Walker et al., 1995). They can be evidenced using DNA dyes, such as propidium iodide (Nicoletti et al., 1991) or acridine orange (AO) (Olivier, 1995), which differentially incorporate into DNA of living and apoptotic cells. Fig. 4F shows histograms of PBMC stimulated overnight with PMA and ionomycin and co-stained with 7-AAD and AO. Interestingly, early apoptotic cells (gated as 7-AADLo cells, dotted line) already incorporate less AO than living cells (gated as 7-AADneg cells, grey line), suggesting that DNA alteration may occur early in the process, as reported previously (Lecoeur et al., 1997; Overbeeke et al., 1998). A moderate but sig- nificant DNA fragmentation in early apoptotic cells was confirmed by in situ nick end labeling following dUTP incorporation (TUNEL assay), while important DNA degradation occurred in late apoptotic cells (Fig. 4F and G).

3.5. Analysis of oncosis, the initial phase of necrosis

Oncosis can be induced by hypotonic shock in distilled water, 3 min of treatment at 56 jC or 4 h of treatment with NaN3, a mitochondrial poison that blocks ATP production and acts at the level of the complex IV of the respiratory chain. In order to define the specificity of both the 7-AAD and the Annexin V/ PI assays, with regard to apoptosis detection, we used them on Jurkat cells induced to die by oncosis following treatment with either NaN3 or H2O (Fig. 5A). As was observed for apoptotic cells, 7-AAD + cells were divided into 7-AADLo and 7-AADHi cells and the Annexin V/PI assay detected a fraction of
Annexin V + /PI — cells, a phenotype considered spe- cific for early apoptotic cells (Vermes et al., 1995). As
observed previously on apoptotic cells, the percen- tage of 7-AADLo cells approximately matched the percentage of oncotic Annexin V + /PI — cells, confir- ming that Annexin V binding is not a specific feature of apoptotic cells (Lecoeur et al., 2001a). The same
observation was made on primary human lympho- cytes submitted to H2O treatment (Fig. 5B). A multi- parametric analysis of oncotic cells was performed on these cells detecting concomitantly the loss of mem- brane integrity by 7-AAD uptake, PS exposure by Annexin V binding and surface CD4 molecule (Fig.

5C). 7-AADLo (oncotic) and 7-AADHi (oncotic necrotic) cells within CD4 T cells are distinguished and analysis of Annexin V staining on these cells shows that oncotic necrotic cells (7-AADHi) expressed higher levels of PS residues (MFI = 805) than did oncotic cells (7-AADLo, MFI = 387). Fig. 5D shows that mitochondrial transmembrane potential, detected by DiOC6(3) staining, is progressively decreased during primary necrosis of lymphocytes. Altogether, these data indicate that following hypotonic shock,
oncotic cells are Annexin V + /PI — , 7-AADLo and DiOC6(3)Lo. This phenotype is similar to that of
apoptotic cells. Therefore, these flow cytometric assays are not able to discriminate between apoptosis and oncosis.

3.6. Proposed strategy to improve flow cytometric quantification of apoptosis

The classical way to quantify apoptotic cells in a given population with either the 7-AAD assay or the Annexin V/PI assay is to consider selectively the so-
called ‘‘early apoptotic cells’’, defined as 7-AADLo or Annexin V + /PI — cells, assuming that 7-AADHi cells and Annexin V + /PI + cells correspond to necrotic cells. However, this principle has been recently chal- lenged by data showing on the one hand that late
apoptotic cells are also 7-AADHi and Annexin V + / PI + and, on the other hand, that ‘‘early apoptotic cells’’ may exhibit the phenotype of oncotic cells (Lecoeur et al., 2001a). In an attempt to analyse the consequences of selective gating of early apoptotic cells during apoptosis quantification, we stimulated PBMC from control donors (resistant to apoptosis) or HIV-infected persons (highly susceptible to apoptosis; Gougeon et al., 1996; Ledru et al., 1998) with either anti-CD3 mAbs (Fig. 6A) or PMA + ionomycin (Fig. 6B) (both stimuli being potent inducers of apoptosis in primed lymphocytes, Fig. 6C), and we quantified apoptosis within the CD8 T cell subset (Fig. 6A) or within IFNg-producing cells (Fig. 6B).
When apoptotic CD8 T cells were quantified in
CD3-stimulated PBMC from control donors, compar- ison of early vs. total apoptotic cells did not show significant differences (2.7% vs. 4.2%), whereas a significant underestimation of apoptotic cells was made when patients’ lymphocytes were analysed (20.9% of early vs. 34.2% of total apoptotic cells)

Fig. 5. Analysis of oncosis in PBMCs and Jurkat cells by flow cytometry. (A) Detection of oncosis and oncotic necrosis in Jurkat cells treated for 4 h with 1% NaN3 or 5 min with distilled water (H2O) by the 7-AAD and Annexin V/PI assays. The percentage of oncotic and oncotic necrotic cells are indicated. (B) Detection of oncotic and oncotic necrotic cells in freshly isolated peripheral blood lymphocytes. PBMCs were isolated upon Ficoll density gradient and submitted to cell adhesion to eliminate monocytes and residual platelets. Lymphocytes were then analysed with both the 7-AAD and the Annexin V/PI assays either directly (ex vivo) or following a 3-min hypotonic shock in distilled water (H2O). The percentage of oncotic and oncotic necrotic cells are indicated. (C) Multiparametric analysis of PBMC submitted to 3-min hypotonic shock in distilled water. Living (7-AADneg), oncotic (7-AADLo) and oncotic necrotic (7-AADHi) CD4 + T lymphocytes were gated on the 7- AAD/CD4 dot plot, and the extent of PS exposure in these subsets was evaluated. MFI of Annexin V staining for each of these subsets is shown.
(D) Detection of the loss of mitochondrial transmembrane potential during oncosis induced by 5-min hyperthermia using 7-AAD/DiOC6(3) double staining. MFI of DiOC6(3) staining is indicated in living, oncotic and oncotic necrotic lymphocytes.

Fig. 6. Consequences of selective gating on early apoptotic cells on apoptosis quantification. (A) PBMCs from a control donor and an HIV- infected subject were incubated overnight with anti-CD3 antibodies. Cells were co-stained with 7-AAD and anti-CD8 mAbs, and analysis was performed on total apoptotic cells or focused on early apoptotic cells. Values in bold indicate the percentage of apoptotic cells within CD8 T cells. (B) PBMCs from the same donors were activated overnight with PMA– ionomycin. Apoptosis in IFNg-producing cells was analysed. Analysis was performed on total apoptotic cells or focused on early apoptotic cells. Values in bold indicate the percentage of apoptotic cells within CD8 T cells. (C) Electron microscopy of PBMCs from an HIV + subject treated with PMA– Iono for 16 h. Two apoptotic cells, with different stages of shrinkage and DNA condensation, are shown.

(Fig. 6A). When PBMCs were stimulated overnight with PMA + ionomycin (a classical way to stimulate polyclonal cytokine synthesis to further enumerate by FACS Th1/Th2 populations, Ledru et al., 1998), the percentage of apoptosis was underestimated both in control and HIV + donors. Indeed, in both samples,

the dead cells were mostly late apoptotic cells (64% and 79% of 7-AAD + cells were 7-AADHi for the control donor and the patient, respectively). Conse- quently, consider that only early apoptotic cells dra- matically underestimated the percentage of dead cells within IFNg-producing cells since they represented

7% vs. 16.7% in control donors and 8.9% vs. 30.1% in HIV + persons (Fig. 6B). The degree of apoptosis within IFNg-producing cells in control and HIV + donor was comparable (7% vs. 8.9%) when early apoptotic cells were considered, whereas it was much higher in the patient vs. control (30.1% vs. 16.7%) when the total number of dead cells was considered. Electron microscopy of PMA + ionomycin-stimulated cells confirmed that almost all of them were dying of apoptosis (Fig. 6C). Altogether, these data underline the complexity of the process of quantification of apoptosis by FACS and, in our experience, discarding late apoptotic cells when analysing primary lympho- cytes submitted to apoptotic stimuli may lead to incorrect interpretations.

4. Discussion

Within the last decade, a growing number of flow cytometric assays for apoptosis detection and quanti- fication have been developed. However, only a few of them allow a multiparametric analysis of apoptotic cells. In this article, we have described the advantages of the 7-AAD assay for the combined study of biochemical, morphological and immunological events in apoptotic cells. This assay was initially set up with primary cultures of lymphoid cells, i.e. murine thy- mocytes and peripheral blood lymphocytes, and it was shown to be capable of detecting early apoptotic cells (7-AADLo cells) (Schmid et al., 1992). Here, we have extended the 7-AAD assay to lymphoid cell lines (CEM, Jurkat cells) and adherent cell lines of hepatic (MMHD3 cells) or cervical (HeLa cells) origin and we have observed different patterns of 7-AAD incor- poration. One of the advantages of the 7-AAD assay is that it permits the phenotyping of apoptotic cells using both murine thymocytes (Schmid et al., 1994a,b) and human PBMCs (Lecoeur et al., 1997). We show here that it can also be used for the co-detection of PS residues on the plasma membrane or transgene- encoded antigens in apoptotic transfected cells. Fur- thermore, this assay can be used for the quantification of lysis of target cells in a cell-mediated cytotoxicity assay (Lecoeur et al., 2001b).
The 7-AAD assay can also be used to detect intracellular molecules such as cytokines or members of the Bcl-2 family (Lecoeur et al., 1998). In this

study, we have shown that mitochondrial alterations, such as the loss of mitochondrial transmembrane potential (Dwm), detected with the cationic dye DiOC6(3), cardiolipin peroxidation detected by the NAO probe or the expression of mitochondrial 7A6 protein with specific mAb, can be observed using 7- AAD-stained cells. This assay is also appropriate for the co-detection of reactive oxygen species (ROS) during dexamethasone-induced apoptosis of immature murine thymocytes (Torres Roca et al., 1995). 7-AAD staining can also be combined with the detection of the active form of intracellular caspase-3 and caspase-
8 using polyclonal antibodies or specific carboxy- fluorescein-labeled caspase inhibitors. Indeed, an active form of both caspases was found to be expressed in early apoptotic cells in PMA + ionomy- cin-stimulated PBMC or in Jurkat cells incubated with CD95-specific mAbs.
Finally, we report that, similar to the TUNEL or the Annexin V/PI assay, the 7-AAD assay is not specific for apoptosis since it also detects primary necrotic cells (Grasl-Kraup et al., 1995; Waring et al., 1999; Bon- neau and Poulin, 2000; Lecoeur et al., 2001a). It is generally accepted that cell death occurs via apoptosis or primary necrosis. However, intermediate cell death stages have been observed in diverse tissues (Portera- Cailliau et al., 1997; Formigli et al., 2000), and both type of cell death can be observed on the same bio- logical samples depending on the dose of the stimulus (Akagi et al., 1993; Palomba et al., 1999; Lecoeur et al., 2001a). It suggests that both type of cell death may represent the extremes of a continuum, the intracellular ATP level being the key factor that determines the phenotype of the dying cell (Leist et al., 1997; Lelli et al., 1998). We show here that similar to early apoptotic cells, oncotic cells (cells at the early stage of primary necrosis) can be detected by weak 7-AAD incorpora- tion (7-AADLo cells). Oncotic necrotic cells show a disrupted plasma membrane, as evidenced by a strong 7-AAD incorporation (7-AADHi cells), similar to 7- AAD incorporation of late apoptotic cells. These observations have two major consequences. First, microscopic examination of dying cells by electron or light microscopy should be performed for each sample in parallel with flow cytometry analysis in order to determine the type of cell death(s) occurring in a given cell sample. If both oncosis and apoptosis are observed, flow cytometric analyses are not appro-

priate. Flow cytometry can be used on samples includ- ing a majority of apoptotic cells, but analysis of these cells should include both early and late (7-AADLo + 7- AADHi) apoptotic cells. The second consequence is that primary necrosis can be studied with the 7-AAD flow cytometric assay. It permits the determination of the relative percentage of oncotic and oncotic necrotic cells and, similar to apoptosis, it can be used in a multiparametric assay to analyse some of the biochem- ical events associated with primary necrosis.

Acknowledgements

We particularly thank Marie Christine Pre´vost for her expertise in electron microscopy and Dr. Eric Ledru for a critical review of the manuscript. This work was supported by grants from the Pasteur Institute, the Agence Nationale de Recherche sur le SIDA (ANRS), Sidaction, the Centre National de la Recherche Scientifique (CNRS) and the European Union (Contract Nos. BMH4-CT 97-2055 and ERB- IC97-0901).

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