Flow Cytometry of Apoptosis

Piotr Pozarowski1, Jerzy Grabarek2, Zbigniew Darzynkiewicz3

1 School of Medicine, Lublin, 2 Pomeranian School of Medicine, Szczecin, Poland, 3 New York Medical College, Valhalla, New York
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.19
DOI:  10.1002/0471142956.cy0719s25
Online Posting Date:  August, 2003
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Abstract

Application of flow cytometry to the study of cell death has three goals: identification and quantification of dead and dying cells; discrimination between apoptotic and necrotic modes of cell death; and elucidation of mechanisms involved in cell death. This massively detailed unit by a pioneer in the field brings together the most common flow cytometric methods for the study of apoptosis, covering a wide variety of apoptotic indices, from loss of membrane potential, caspase activation, and phosphatidyl exposure to DNA fragmentation and tissue transglutaminase activation. The authors also present their recently developed protocol, analogous to the FLICA approach for caspases, for the detection of serine proteases (‘serpases’). The protocols are accompanied by extensive commentary discussion of applicability, strategic planning, problems, and pitfalls, plus a comprehensive list of references.

Keywords: flow cytometry; apoptosis; membrane potential; caspase activation; annexin V; serine proteases; DNA fragmentation; FLICA; TUNEL

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Mitochondrial Transmembrane Potential (Δψm) Measured by Rhodamine 123 or DiOC6(3) Fluorescence
  • Basic Protocol 2: Immunocytochemical Detection of Activated Caspases by Zenon Technology
  • Basic Protocol 3: Detection of Apoptotic Cells Using Fluorochrome‐Labeled Inhibitors of Caspases (FLICAs)
  • Basic Protocol 4: Determination of Poly(ADP‐Ribose) Polymerase (PARP) Cleavage
  • Basic Protocol 5: Annexin V Binding
  • Basic Protocol 6: DNA Fragmentation: Detection of Cells with Fractional (Sub‐G1) DNA Content Using PI
  • Alternate Protocol 1: DNA Fragmentation: Detection of Cells with Fractional (“Sub‐G1”) DNA Content Using DAPI
  • Basic Protocol 7: DNA Fragmentation: Detection of DNA Strand Breaks (TUNEL Assay)
  • Basic Protocol 8: Detection of Tissue Transglutaminase Activation by Cell Resistance to Detergents
  • Alternate Protocol 2: Detection of TGase 2 Activation by Fluoresceinated Cadaverine (F‐CDV) Binding
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Mitochondrial Transmembrane Potential (Δψm) Measured by Rhodamine 123 or DiOC6(3) Fluorescence

  Materials
  • Cells of interest in appropriate complete culture medium
  • 10 µM rhodamine 123 (R123; see recipe) or 10 µM DiOC 6(3) (see recipe for 0.1 mM stock solution) or 0.2 mM JC‐1 stock solution (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 1 mg/ml propidium iodide (PI; Molecular Probes) in distilled water; store at 4°C in the dark
  • 12 × 75–mm tubes suitable for flow cytometer
  • Flow cytometer with 488‐nm excitation and filters for collection of green, orange, and red fluorescence

Basic Protocol 2: Immunocytochemical Detection of Activated Caspases by Zenon Technology

  Materials
  • Cells of interest (see appendix 3B for culture techniques), both untreated (control) and induced to apoptosis (e.g., exponentially growing HL‐60 cells incubated 2 to 4 hr with 0.15 µM camptothecin)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Fixatives:
    • 1% (v/v) methanol‐free formaldehyde (Polysciences) in PBS, 0° to 5°C
    • 4% (v/v) methanol‐free formaldehyde (Polysciences) in PBS, room temperature
    • 70% (v/v) ethanol, –20°C
  • Rinse solution (see recipe)
  • Primary antibody: cleaved (activated) caspase‐3 antibody (Cell Signaling Technology, cat. no. 9661)
  • Zenon Alexa Fluor 488 rabbit IgG labeling kit (Molecular Probes, cat. no. Z‐25302)
  • 10% (v/v) Triton X‐100 in PBS
  • DNA staining solution with PI (see recipe)
  • 12 × 75–ml tubes suitable for use on flow cytometer
  • Flow cytometer with 488‐nm excitation and filters for collection of green and red fluorescence

Basic Protocol 3: Detection of Apoptotic Cells Using Fluorochrome‐Labeled Inhibitors of Caspases (FLICAs)

  Materials
  • Cells of interest (see appendix 3B for culture techniques)
  • Medium supplemented with 10% (v/v) serum or 1% (w/v) serum albumin
  • FLICA kit (Immunochemistry Technologies) containing:
    • FAM‐VAD‐FMK reagent (see recipe)
    • Fixative
    • Hoechst stain
  • Rinse solution: 1% (w/v) BSA in PBS ( appendix 2A)
  • 1 mg/ml propidium iodide (PI; Molecular Probes) in distilled water; store at 4°C in the dark
  • 12 × 75–ml tubes suitable for use on flow cytometer
  • Flow cytometer with 488‐nm excitation and filters for collection of green and red fluorescence

Basic Protocol 4: Determination of Poly(ADP‐Ribose) Polymerase (PARP) Cleavage

  Materials
  • Cells of interest
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 1% methanol‐free formaldehyde (Polysciences) in PBS ( appendix 2A)
  • 70% ethanol
  • 0.25% (v/v) Triton X‐100 (Sigma) in PBS ( appendix 2A); store at 4°C
  • PBS/BSA solution: 1% (w/v) bovine serum albumin (Sigma) in PBS; store at 4°C
  • Anti‐PARP p85 antibody (Promega anti‐PARP‐85 fragment, rabbit polyclonal)
  • Fluorescein‐conjugated anti‐rabbit immunoglobulin antibody (Dako)
  • 1 mg/ml propidium iodide (PI; Molecular Probes) in distilled water; store at 4°C in the dark
  • RNase solution ( appendix 2A)
  • 12 × 75–mm centrifuge tubes suitable for use on the flow cytometer
  • Pasteur pipets
  • Flow cytometer with 488‐nm excitation and filters for collection of green and red fluorescence

Basic Protocol 5: Annexin V Binding

  Materials
  • Cells of interest
  • Fluorescein‐conjugated annexin V (see recipe) in binding buffer (see recipe)
  • 1 mg/ml propidium iodide (PI; Molecular Probes) in distilled water; store at 4°C in the dark
  • Flow cytometer with 488‐nm excitation and filters for collection of green and red fluorescence

Basic Protocol 6: DNA Fragmentation: Detection of Cells with Fractional (Sub‐G1) DNA Content Using PI

  Materials
  • Cells of interest
  • PBS ( appendix 2A)
  • 70% ethanol
  • DNA extraction buffer (see recipe)
  • DNA staining solution with PI (see recipe)
  • 12 × 75–mm tubes suitable for use on the flow cytometer
  • Flow cytometer with 488‐nm excitation and filter for collection of red fluorescence

Alternate Protocol 1: DNA Fragmentation: Detection of Cells with Fractional (“Sub‐G1”) DNA Content Using DAPI

  • DNA staining solution with DAPI (see recipe)
  • Flow cytometer equipped with UV excitation and filter for collection of blue fluorescence

Basic Protocol 7: DNA Fragmentation: Detection of DNA Strand Breaks (TUNEL Assay)

  Materials
  • Cells of interest
  • 1% (v/v) methanol‐free formaldehyde (Polysciences) in PBS ( appendix 2A), pH 7.4 (primary fixative)
  • PBS ( appendix 2A)
  • 70% ethanol (secondary fixative)
  • 5× TdT reaction buffer (see recipe)
  • 2 mM BrdUTP (Sigma) in 50 mM Tris·Cl, pH 7.5
  • TdT in storage buffer (both from Roche Diagnostics), 25 U in 1 µl
  • 10 mM cobalt chloride (CoCl 2; Roche Diagnostics)
  • Rinsing buffer: PBS with 0.1% (v/v) Triton X‐100 and 0.5% (w/v) BSA
  • FITC‐conjugated anti‐BrdU MAb in PBS ( appendix 2A; see recipe)
  • PI staining buffer: PBS with 5 µg/ml PI and 200 µg/ml DNase‐free RNase
  • Flow cytometer with 488‐nm excitation and filters for collection of green and red fluorescence

Basic Protocol 8: Detection of Tissue Transglutaminase Activation by Cell Resistance to Detergents

  Materials
  • Cells of interest
  • DAPI/sulforhodamine 101/detergent solution (see recipe)
  • Flow cytometer equipped with UV excitation and filters for collection of blue and red fluorescence

Alternate Protocol 2: Detection of TGase 2 Activation by Fluoresceinated Cadaverine (F‐CDV) Binding

  Materials
  • Fluoresceinated cadaverine solution (F‐CDV; see recipe)
  • Cells of interest
  • 100% methanol
  • DNA staining solution with PI (see recipe)
  • Flow cytometer with 488‐nm excitation and filters for collection of green and red fluorescence
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Figures

Videos

Literature Cited

Literature Cited
   Alnemri, E.S., Livingston, D.I., Nicholson, D.W., Salvesen, G., Thornberry, N.A., Wong, W.W., and Yuan, J. 1996. Human ICE/CED‐4 protease nomenclature. Cell 87:171‐173.
   Arends, M.J., Morris, R.G., and Wyllie, A.H. 1990. Apoptosis: The role of endonuclease. Am. J. Pathol. 136:593‐608.
   Bedner, E., Burfeind, P., Gorczyca, W., Melamed, M.R., and Darzynkiewicz, Z. 1997. Laser scanning cytometry distinguishes lymphocytes, monocytes and granulocytes by differences in their chromatin structure. Cytometry 29:191‐196.
   Bedner, E., Li, X., Gorczyca, W., Melamed, M.R., and Darzynkiewicz, Z. 1999. Analysis of apoptosis by laser scanning cytometry. Cytometry 35:181‐195.
   Blagosklonny, M.V. 2000. Cell death beyond apoptosis. Leukemia 14:1502‐1508.
   Budihardjo, I., Oliver, H., Lutter, M., and Luo, X. 1999. Biochemical pathways of caspase activation during apoptosis. Annu. Rev. Cell Dev. Biol. 15:269‐290.
   Catchpoole, D.R. and Stewart, B.W. 1993. Etoposide‐induced cytotoxicity in two human T‐cell leukemic lines. Delayed loss of membrane permeability rather than DNA fragmentation as an indicator of programmed cell death. Cancer Res. 53:4287‐4296.
   Collins, R.J., Harmon, B.V., Gobe, G.C., and Kerr, J.F.R. 1992. Internucleosomal DNA cleavage should not be the sole criterion for identifying apoptosis. Int. J. Radiat. Biol. 61:451‐453.
   Cossarizza, A. and Salvioli, S. 2001. Analysis of mitochondria during cell death. Meth. Cell Biol. 63:467‐486.
   Cossarizza, A., Kalashnikova, G., Grassilli, E., Chiappelli, F., Salvioli, S., Capri, M., Barbieri, D., Troiano, L., Monti, D., and Franceschi, C. 1994. Mitochondrial modifications during rat thymocyte apoptosis: A study at a single cell level. Exp. Cell Res. 214:323‐330.
   Darzynkiewicz, Z., Staiano‐Coico, L., and Melamed, M.R. 1981. Increased mitochondrial uptake of rhodamine 123 during lymphocyte stimulation. Proc. Natl. Acad. Sci. U.S.A. 78:2383‐2387.
   Darzynkiewicz, Z., Traganos, F., Staiano‐Coico, L., Kapuscinski, J., and Melamed, M.R. 1982. Interactions of rhodamine 123 with living cells studied by flow cytometry. Cancer Res. 42:799‐806.
   Darzynkiewicz, Z., Traganos, F., and Kimmel, M. 1987. Assay of cell cycle kinetics by multivariate flow cytometry using the principle of stathmokinesis. In Techniques in Cell Cycle Analysis (J.W.A. Gray and Z.A. Darzynkiewicz, eds.) pp. 291‐336. Humana Press, Totowa, N.J.
   Darzynkiewicz, Z., Bruno, S., Del Bino, G., Gorczyca, W., Hotz, M.A., Lassota, P., and Traganos, F. 1992. Features of apoptotic cells measured by flow cytometry. Cytometry 13:795‐808.
   Darzynkiewicz, Z., Li, X., and Gong, J. 1994. Assays of cell viability. Discrimination of cells dying by apoptosis. Meth. Cell Biol. 41:16‐39.
   Darzynkiewicz, Z., Juan, G., Li, X., Murakami, T., and Traganos, F. 1997. Cytometry in cell necrobiology: Analysis of apoptosis and accidental cell death (necrosis). Cytometry 27:1‐20.
   Darzynkiewicz, Z., Bedner, E., Li, X., Gorczyca, W., and Melamed, M.R. 1999. Laser scanning cytometry. A new instrumentation with many applications. Exp. Cell Res. 249:1‐12.
   Deng, Y., Lin, Y., and Wu, X. 2002. TRAIL‐induced apoptosis requires Bax‐dependent mitochondrial release of Smac/DIABLO. Genes Dev. 16:33‐45.
   de Murcia, G. and Menissier‐de Murcia, J.M. 1994. Poly(ADP‐ribose) polymerase: A molecular nick sensor. Trends Biochem. Sci. 19:172‐176.
   Earnshaw, W.C., Martins, L.M., and Kaufmann, S.H. 1999. Mammalian caspases: Structure, activation, substrates, and functions during apoptosis. Annu. Rev. Biochem. 68:383‐424.
   Fadok, V.A., Voelker, D.R., Campbell, P.A., Cohen, J.J., Bratton, D.L., and Henson, P.M. 1992. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148:22‐29.
   Ferlini, C., Scambia, G., and Fattorossi, A. 1998. Is chloromethyl‐X‐rosamine useful in measuring mitochondrial transmembrane potential? Cytometry 31:74‐79.
   Fesus, L., Thomazy, V., and Falus, A. 1987. Induction and activation of tissue transglutaminase during programmed cell death. FEBS Lett. 224:104‐108.
   Finucane, D.M., Waterhouse, N.J., Amaranto‐Mendes, G.P., Cotter, T.G., and Green, D.R. 1999. Collapse of the inner mitochondrial transmembrane potential is not required for apoptosis of HL‐60 cells. Exp. Cell Res. 251:166‐174.
   Gilmore, K. and Wilson, M. 1999. The use of chloromethyl‐X‐rosamine (Mitotracker Red) to measure loss of mitochondrial membrane potential in apoptotic cells is incompatible with cell fixation. Cytometry 36:355‐358.
   Gong, J., Traganos, F., and Darzynkiewicz, Z. 1994. A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. Anal. Biochem. 218:314‐319.
   Gorczyca, W., Bruno, S., Darzynkiewicz, R., Gong, J., and Darzynkiewicz, Z. 1992. DNA strand breaks occurring during apoptosis: Their early in situ detection by the terminal deoxynucleotidyl transferase and nick translation assays and prevention by serine protease inhibitors. Int. J. Oncol. 1:639‐648.
   Gorczyca, W., Bigman, K., Mittelman, A., Ahmed, T., Gong, J., Melamed, M.R., and Darzynkiewicz, Z. 1993. Induction of DNA strand breaks associated with apoptosis during treatment of leukemias. Leukemia 7:659‐670.
   Gorman, A.M., Hirt, U.A., Zhivotovsky, B., Orrenius, S., and Ceccatelli, S. 1999. Application of a fluorometric assay to detect caspase activity in thymus tissue undergoing apoptosis in vivo. J. Immunol. Methods 226:43‐48.
   Grabarek, J., Ardelt, B., Kunicki, J., and Darzynkiewicz, Z. 2002. Detection of in situ activation of transglutaminase during apoptosis: Correlation with the cell cycle phase by multiparameter flow and laser scanning cytometry. Cytometry 49:83‐89.
   Haugland, R.P. 2002. Handbook of Fluorescent Probes and Research Chemicals. Ninth Edition. Molecular Probes, Eugene, OR.
   Hotz, M.A., Traganos, F., and Darzynkiewicz, Z. 1992. Changes in nuclear chromatin related to apoptosis or necrosis induced by the DNA topoisomerase II inhibitor fostriecin in MOLT‐4 and HL‐60 cells are revealed by altered DNA sensitivity to denaturation. Exp. Cell Res. 201:184‐191.
   Hotz, M.A., Gong, J., Traganos, F., and Darzynkiewicz, Z. 1994. Flow cytometric detection of apoptosis: Comparison of the assays of in situ DNA degradation and chromatin changes. Cytometry 15:237‐244.
   Hug, H., Los, M., Hirt, W., and Debatin, K.M. 1999. Rhodamine 110‐linked amino acids and peptides as substrates to measure caspase activity upon apoptosis induction in intact cells. Biochemistry 38:13906‐13911.
   Johnson, L.V., Walsh, M.L., and Chen, L.B. 1980. Localization of mitochondria in living cells with rhodamine 123. Proc. Natl. Acad. Sci. U.S.A. 77:990‐994.
   Kamentsky, L.A. 2001. Laser scanning cytometer. Meth. Cell Biol. 63:51‐87.
   Kaufmann, S.H., Desnoyers, S., Ottaviano, Y., Davidson, N.E., and Poirier, G.G. 1993. Specific proteolytic cleavage of poly(ADP‐ribose) polymerase: An early marker of chemotherapy‐induced apoptosis. Cancer Res. 53:3976‐3985.
   Keiji, J.F., Bell‐Prince, C., and Steinkamp, J.A. 2000. Staining of mitochondrial membranes with 10‐nonyl acridine orange, MitoFluor Green, and MitoTracker Green is affected by mitochondrial membrane potential altering drugs. Cytometry 39:203‐210.
   Kerr, J.F.R., Wyllie, A.H., and Curie, A.R. 1972. Apoptosis: A basic biological phenomenon with wide‐ranging implications in tissue kinetics. Br. J. Cancer 26:239‐257.
   Knapp, P.E., Bartlett, W.P., Williams, L.A., Yamada, M., Ikenaka, K., and Skoff, R.P. 1999. Programmed cell death without DNA fragmentation in the jimpy mouse: Secreted factors can enhance survival. Cell Death Differ. 6:136‐145.
   De Kockx, M.M., Meyer, G.R., Muhring, J., Jacob, W., Bult, H., and Herman, A.G. 1998. Apoptosis and related proteins in different stages of human atherosclerotic plaques. Circulation 97:2307‐2315.
   Komoriya, A., Packard, B.Z., Brown, M.J., Wu, M.L., and Henkart, P.A. 2000. Assessment of caspase activities in intact apoptotic thymocytes using cell‐permeable caspase substrates. J.Exp.Med. 191:1819‐1828.
   Koopman, G., Reutelingsperger, C.P.M., Kuijten, G.A.M., Keehnen, R.M.J., Pals, S.T., and van Oers, M.H.J. 1994. Annexin V for flow cytometric detection of phosphatidylserine expression of B cells undergoing apoptosis. Blood 84:1415‐1420.
   Kroemer, G. 1998. The mitochondrion as an integrator/coordinator of cell death pathways. Cell Death Differ. 5:547‐548.
   Lajemi, M., Demignot, S., and Adolphe, M. 1998. Detection and characterization, using fluoresceincadaverine, of amine acceptor substrates accessible to active transglutaminase expressed by rabbit articular chondrocytes. Histochem. J. 30:499‐508.
   Lazebnik, Y.A., Kaufmann, S.H., Desnoyers, S., Poirier, G.G., and Earnshaw, W.C. 1994. Cleavage of poly(ADP‐ribose) polymerase by proteinase with properties like ICE. Nature 371:346‐347.
   Li, X., and Darzynkiewicz, Z. 1995. Labelling DNA strand breaks with BdrUTP. Detection of apoptosis and cell proliferation. Cell Prolif. 28:571‐579.
   Li, X., and Darzynkiewicz, Z. 2000. Cleavage of poly(ADP‐ribose) polymerase measured in situ in individual cells: Relationship to DNA fragmentation and cell cycle position during apoptosis. Exp. Cell Res. 255:125‐132.
   Li, X., Traganos, F., Melamed, M.R., and Darzynkiewicz, Z. 1995. Single‐step procedure for labeling DNA strand breaks with fluorescein‐ or BODIPY‐conjugated deoxynucleotides: Detection of apoptosis and bromodeoxyuridine incorporation. Cytometry 20:172‐180.
   Li, X., Melamed, M.R., and Darzynkiewicz, Z. 1996. Detection of apoptosis and DNA replication by differential labeling of DNA strand breaks with fluorochromes of different color. Exp. Cell Res. 222:28‐37.
   Li, X., Du, L., and Darzynkiewicz, Z. 2000. During apoptosis of HL‐60 and U‐937 cells caspases are activated independently of dissipation of mitochondrial electrochemical potential. Exp. Cell Res. 257:290‐297.
   Liu, X., Kim, C.N., Yang, J., Jemmerson, R., and Wang, X. 1996. Induction of apoptotic program in cell‐free extracts: Requirements for dATP and cytochrome c. Cell 86:147‐157.
   Liu, J., Bhalgat, M., Zhang, C., Diwu, Z., Hoyland, B., and Klaubert, D.H. 1999. Fluorescent molecular probes V: A sensitive caspase‐3 substrate for fluorometric assays. Bioorg. Med. Chem. Lett. 9:3231‐3236.
   Majno, G. and Joris, I. 1995. Apoptosis, oncosis, and necrosis. An overview of cell death. Am. J. Pathol. 146:3‐16.
   Marguet, D., Luciani, M.‐F., Moynault, A., Williamson, P., and Chimini, G. 1999. Engulfment of apoptotic cells involves the redistribution of membrane phosphatidylserine on phagocyte and prey. Nature Cell Biol. 1:454‐456.
   Melino, E. and Piacentini, M. 1998. “Tissue” transglutaminase in cell death: A downstream or multifunctional upstream effector? FEBS Lett. 430:59‐63.
   Nagata, S. 2000. Apoptotic DNA fragmentation. Exp. Cell Res. 256:12‐18.
   Nicoletti, I., Migliorati, G., Pagliacci, M.C., Grignani, F., and Riccardi, C. 1991. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods 139:271‐280.
   Oberhammer, F., Wilson, J.M., Dive, C., Morris, I.D., Hickman, J.A., Wakeling, A.E., Walker, P.R., and Sikorska, M. 1993. Apoptotic death in epithelial cells: Cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J. 12:3679‐3684.
   Ormerod, M.G. 1998. The study of apoptotic cells by flow cytometry. Leukemia 12:1013‐1025.
   Ormerod, M.G., O'Neill, C.F., Robertson, D., and Harrap, K.R. 1994. Cisplatin induced apoptosis in a human ovarian carcinoma cell line without a concomitant internucleosomal degradation of DNA. Exp. Cell Res. 211:231‐237.
   Ormerod, M.G., Cheetham, F.P.M., and Sun, X.‐M. 1995. Discrimination of apoptotic thymocytes by forward light scatter. Cytometry 21:300‐304.
   Petit, J.M., Ratinaud, M.H., Cordelli, E., Spano, M., and Julien, R. 1995. Mouse testis cell sorting according to DNA and mitochondrial changes during spermatogenesis. Cytometry 19:304‐312.
   Poot, M., Gibson, L.L., and Singer, V.L. 1997. Detection of apoptosis in live cells by MitoTracker Red CMXRos and SYTO dye flow cytometry. Cytometry 27:358‐364.
   Pozarowski, P., Halicka, D.H., and Darzynkiewicz, Z. 2003. The NK‐κB inhibitor sesquiterpene parthenolide induces concurrently atypical apoptosis and cell necrosis: Difficulties in identification of dead cells in such cultures. Cytometry. In press.
   Ratinaud, M.H., Leprat, P., and Julien, R. 1988. In situ cytometric analysis of nonyl acridine orange‐stained mitochondria from splenocytes. Cytometry 9:206‐212.
   Sallman, F.R., Bourassa, S., Saint‐Cyr, J., and Poirier, G.G. 1997. Characterization of antibodies specific for the caspase cleavage site on poly(ADP‐ribose) polymerase: Specific detection of apoptotic fragments and mapping of the necrotic fragments of poly(ADP‐ribose) polymerase. Biochem. Cell Biol. 75:451‐458.
   Scorrano, L., Petronilli, V., Colonna, R., Di Lisa, F., and Bernard, P. 1999. Chloromethyltetramethylrosamine (Mitotracker Orange) induces the mitochondrial permeability transition and inhibits respiratory complex I. Implications for the mechanism of cytochrome c release. J. Biol. Chem. 274:24657‐24663.
   Shi, Y. 2002. Mechanisms of caspase activation and inhibition during apoptosis. Molec. Cell 9:459‐470.
   Smolewski, P., Bedner, E., Du, L., Hsieh, T.‐C., Wu, J.M., Phelps, D.J., and Darzynkiewicz, Z. 2001. Detection of caspases activation by fluorochrome‐labeled inhibitors: Multiparameter analysis by laser scanning cytometry. Cytometry 44:73‐82.
   Susin, S.A., Zamzani, N., Larochette, N., Dallaporta, B., Marzo, I., Brenner, C., Hirsch, T., Petit, P.X., Geuskens, M., and Kroemer, G. 1997. A cytofluorometric assay of nuclear apoptosis induced in a cell‐free system: Application to ceramide‐induced apoptosis. Exp. Cell Res. 236:397‐403.
   Swat, W., Ignatowicz, L., and Kisielow, P. 1981. Detection of apoptosis of immature CD4+8+ thymocytes by flow cytometry. J. Immunol. Methods 137:79‐87.
   Telford, W.G., Komoriya, A., and Packard, B.Z. 2002. Detection of localized caspase activity in early apoptotic cells by laser scanning cytometry. Cytometry 47:81‐88.
   Umansky, S.R., Korol, B.A., and Nelipovich, P.A. 1981. In vivo DNA degradation in the thymocytes of gamma‐irradiated or hydrocortisone‐treated rats. Biochim. Biophys. Acta 655:9‐17.
   van Engeland, M., Nieland, L.J.W., Ramaekers, F.C.S., Schutte, B., and Reutelingsperger, P.M. 1998. Annexin V–affinity assay: A review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 31:1‐9.
   Vermes, I., Haanen, C., and Reutelingsperger, C. 2000. Flow cytometry of apoptotic cell death. J. Immunol. Methods 243:167‐190.
   Waggoner, A.S. 1979. Dye indicators of membrane potential. Annu. Rev. Biophys. Bioeng. 8:47‐68.
   Yang, J., Liu, X., Bhalla, K., Ibrado, A.M., Cai, J., Peng, T.I., Jones, D.P., and Wang, X. 1997. Prevention of apoptosis by Bcl‐2: Release of cytochrome c from mitochondria blocked. Science 275:1129‐1132.
   Zamzani, N., Brenner, C., Marzo, I., Susin, S.A., and Kroemer, G. 1998. Subcellular and submitochondrial mode of action of Bcl‐2‐like oncoproteins. Oncogene 16:2265‐2282.
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