Abstract
Activation of caspases is a hallmark of apoptosis. Several methods, therefore, were developed to identify and count the frequency of apoptotic cells based on the detection of caspases activation. The method described in this chapter is based on the use of f luorochrome-labeled inhibitors of caspases (FLICA) applicable to fluorescence microscopy, and flow- and image-cytometry. Cell-permeant FLICA reagents tagged with carboxyfluorescein or sulforhodamine when applied to live cells in vitro or in vivo, exclusively label cells that are undergoing apoptosis. The FLICA labeling methodology is simple, rapid, robust, and can be combined with other markers of cell death for multiplexed analysis. Examples are presented on FLICA use in combination with a vital stain (propidium iodide), detection of the loss of mitochondrial electrochemical potential, and exposure of phosphatidylserine on the outer surface of plasma cell membrane using Annexin V fluorochrome conjugates. Following cell fixation and stoichiometric staining of cellular DNA, FLICA binding can be correlated with DNA ploidy, cell cycle phase, DNA fragmentation, and other apoptotic events whose detection requires cell permeabilization. The “time window” for the detection of apoptosis with FLICA is wider compared to that with the Annexin V binding, making FLICA a preferable marker for the detection of early phase apoptosis and more accurate for quantification of apoptotic cells.
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References
Salvesen G.S., and Riedl, S.J. (2008) Caspase mechanisms. Adv. Exp. Med. Biol., 615, 13–23.
Kroemer, G., Galluzzi, L., Vandenabeele, P., Abrams, J., Alnemri, E.S., Baehrecke, E.H., Blagosklonny, M.V., El-Deiry, W.S., Golstein, P., Green, D.R., Hengartner, M., Knight, R.A., Kumar, S., Lipton, S.A., Malorni, W., Nunez, G., Peter, M.E., Tschopp, J., Yuan,J., Piacentini, M., Zhivotovski, B., and Melino, G. (2008) Classification of cell death: recommendations of the nomenclature committee on cell death 2009. Cell Death Differ., 16, 3–11.
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.
Nicholson, D.W. (1999) Caspase structure, proteolytic substrates and function during apoptotic cell death. Cell Death Differ., 6, 1028–1042.
Komoriya, A., Packard, B.Z., Brown, M.J., Wu, M.L., and Henkart, P.A. (2000) Assess-ment of caspase activities in intact apoptotic thymocytes using cell-permeable fluorogenic caspase substrates. J. Exp. Med., 191, 1819–1828.
Lee, B.W., Johnson, G.L., Hed, S.A., Darzynkiewicz, Z., Talhouk, J.W., and Mehrota, S. (2003) DEVDase detection in intact apoptotic cells using the cell permeant fluorogenic substrate, (z-DEVD)2-cresyl violet. BioTechniques, 35, 1080–1085.
Pozarowski, P., Huang, X., Halicka D.H., Lee, B., Johnson, G., and Darzynkiewicz, Z. (2003) Interactions of fluorochrome-labeled caspase inhibitors with apoptotic cells. A caution in data interpretation. Cytometry A, 55A, 50–60.
Huang, X., Okafuji, M., Traganos, F., Luther, E., Holden, E., and Darzynkiewicz, Z. (2004) Assessment of histone H2AX phosphorylation induced by DNA topoisomerase I and II inhibitors topotecan and mitoxantrone and by DNA crosslinking agent cisplatin. Cytometry A, 58A, 99–110.
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.
Bedner, E., Smolewski, P., Amstad, P., and Darzynkiewicz, Z. (2000) Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation. Exp. Cell Res., 260, 308–313.
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.
Darzynkiewicz, Z., and Barnard, E.A. (1966) Specific proteases of mast cells. Nature, 212, 1198–1203.
Smolewski, P., Grabarek, J., Lee, B.W., Johnson, G.L., and Darzynkiewicz, Z. (2002) Kinetics of HL-60 cell entry to apoptosis during treatment with TNF-α or camptothecin assayed by the stathmo-apoptosis method. Cytometry, 47, 143–149.
Smolewski, P., Grabarek, J., Phelps, D.J., and Darzynkiewicz, Z. (2001) Stathmo-apoptosis: arresting apoptosis by fluorochrome-labeled inhibitor of caspases. Int. J. Oncol., 19, 657–663.
Thornberry, N.A., Rano, T.A., Peterson, E.P., Rasper, D.M., Timkey, T., Garcia-Calvo, M., Houtzager, V.M., Nordstrom, P.A., Roy, S., Valliancourt, J.P., Chapman, K.T., and Nicholson, D.W. (1997) A combinatorial approach defines specificities of members of the caspase family and granzyme B. J. Biol. Chem., 272, 17907–17911.
Darzynkiewicz, Z., and Pozarowski, P. (2007) All that glitters is not gold: all that binds FLICA is not caspase. A caution in data interpretation – and new opportunities. Cytometry A, 71A, 536–537.
Pendergrass, W., Wolf, and Poot, M. (2004) Efficacy of MitoTracker Green and CMXRosamine to measure changes in mitochondrial membrane potentials in living cells and tissues. Cytometry, 61, 162–169.
Koopman, G., Reutelingsperger, C.P.M., Kuijten, G.A.M., Kechnen, R.M.J., Pals, S.T., and van Oers, M.H.H. (1994) Annexin V for flow cytometric detection of phosphatidylderine expression of B cells undergoing apoptosis. Blood, 84, 1415–1420.
Pozarowski, P., Holden, E., and Darzynkiewicz, Z. (2005) Laser scanning cytometry. Principles and applications. Methods Mol. Biol., 319, 165–192.
Darzynkiewicz, Z., Bedner, E., and Traganos, F. (2001) Difficulties and pitfalls in analysis of apoptosis. Methods Cell Biol., 63, 527–559.
Wlodkowic, D., Skommer, J., Hillier, C., and Darzynkiewicz, Z. (2008) Multiparameter detection of apoptosis using red-excitable SYTO probes. Cytometry A, 73A, 563–569.
Griffin R.J., Williams, B.W., Bischof, J.C., Olin, M., Johnson, G.L., and Lee, B.W. (2007) Use of fluorescently labeled poly-caspase inhibitor for in vivo detection of apoptosis related to vascular-targeting agent arsenic trioxide for cancer therapy. Technol. Cancer Res. Treat., 6, 651–654.
Lee, B.W., Olin, M.R., Johnson, G.L., and Griffin, R.J. (2008) In vitro and in vivo apoptosis detection using membrane permeant fluorescent-labeled inhibitors of caspases. Methods Mol. Biol., 414, 109–135.
Tsai, Y.C., Mendoza, A., Mariano, J.M., Zhou, M., Kostova, Z., Chen, B., Veenstra, T., Hewitt, S.M., Helman, L.J., Khanna, C., and Weissman, A.M. (2007) The ubiquitin ligase gp78 promotes sarcoma metastasis by targeting KAI1 for degradation. Nat. Med., 13, 1504–1509.
Cursio, R., Colosetti, P., Auberger, P., and Gugenheim, J. (2008) Liver apoptosis following normothermic ischemia-reperfusion: in vivo evaluation of caspase activity by FLIVO assay in rats. Transplant. Proc., 40, 2038–2041.
Cursio, R., Miele, C., Filippa, N., Colosetti, P., Auberger, P., Van Obberghen, E., and Gugenheim, J. (2009) Tyrosine phosphorylation of insulin receptor substrates during ischemia/reperfusion-induced apoptosis in rat liver. Langenbecks Arch. Surg., 394, 123–131.
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This study was supported by NCI grant RO1 28704.
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Darzynkiewicz, Z., Pozarowski, P., Lee, B.W., Johnson, G.L. (2011). Fluorochrome-Labeled Inhibitors of Caspases: Convenient In Vitro and In Vivo Markers of Apoptotic Cells for Cytometric Analysis. In: Didenko, V. (eds) DNA Damage Detection In Situ, Ex Vivo, and In Vivo. Methods in Molecular Biology, vol 682. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-409-8_9
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DOI: https://doi.org/10.1007/978-1-60327-409-8_9
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