Skip to main content
SpringerLink
Log in
Book cover

Apoptosis and Cancer pp 109–135Cite as

In Vitro and In Vivo Apoptosis Detection Using Membrane Permeant Fluorescent-Labeled Inhibitors of Caspases

  • Protocol

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 414))

Summary

Apoptosis detection methodology is an ever evolving science. The caspase family of cysteine proteases plays a central role in this environmentally conserved mechanism of regulated cell death. New methods that allow for the improved detection and monitoring of the apoptosis-associated proteases are key for further advancement of our understanding of apoptosis-mediated disease states such as cancer and Alzheimer’s disease. From the use of membrane permeant fluorescent-labeled inhibitors of caspases (FLICA) probe technology, we have demonstrated their successful use as tools in the detection of apoptosis activity within the in vitro and in vivo research setting. In this chapter, we provide detailed methods for performing in vitro apoptosis detection assays in whole living cells, using flow cytometry, and 96-well fluorescence plate reader analysis methods. Furthermore, novel flow cytometry-based cytotoxicity assay methods, which incorporate the FLICA probe for early apoptosis detection, are described. Inclusion of this sensitive apoptosis detection probe component into the flow-based cytotoxicity assay format results in an extremely sensitive cytotoxicity detection mechanism. Lastly, in this chapter, we describe the use of the FLICA probe for the in vivo detection of tumor cell apoptosis in mice and rats. These early stage in vivo-type assays show great potential for whole animal apoptosis detection research.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Kerr, J.F.R., Wyllie, A.H., and Currie, A.R. (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239–257.

    Article  CAS  PubMed  Google Scholar 

  2. Wyllie, A.H., Kerr, J.F.R., and Currie, A.R. (1981) Cell death: the significance of apoptosis. Int. Rev. Cytol. 68, 251–306.

    Article  Google Scholar 

  3. 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.

    Article  CAS  PubMed  Google Scholar 

  4. Nicholson, D.W. (1999) Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ. 6, 1028–1042.

    Article  CAS  PubMed  Google Scholar 

  5. Heemels, M.T. (ed.) (2000) Nature insight apoptosis. Nature 407, 770–816.

    Google Scholar 

  6. Degterev, A., Boyce, M., and Yuan, J. (2003) A decade of caspases. Oncogene 22, 8543–8567.

    Article  CAS  PubMed  Google Scholar 

  7. Fink, S.L. and Cookson, B.T. (2005) Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells. Infect. Immun. 73, 1907–1916.

    Article  CAS  PubMed  Google Scholar 

  8. Lavrik, I.N., Golks, A., and Krammer, P.H. (2005) Caspases: pharmacological manipulation of cell death. J. Clin. Invest. 115, 2665–2672.

    Article  CAS  PubMed  Google Scholar 

  9. Thornberry, N.A. and Lazebnik, Y. (1998) Caspases: enemies within. Science 281, 1312–1316.

    Article  CAS  PubMed  Google Scholar 

  10. Thornberry, N.A. et al. (1992) A novel heterodimeric cysteine protease is required for interleukin-1UPbeta processing in monocytes. Nature 356, 768–774.

    Article  CAS  PubMed  Google Scholar 

  11. Thornberry, N.A. et al. (1997) A combinatorial approach defines specificities of members of the caspase family and granzyme B. J. Biol. Chem. 272, 17907–17911.

    Article  CAS  PubMed  Google Scholar 

  12. Fuentes-Prior, P. and Salvesen, G.S. (2004) The protein structures that shape caspase activity, specificity, activation and inhibition. Biochem. J. 384, 201–232.

    Article  CAS  PubMed  Google Scholar 

  13. Sarin, A., Wu, M.L., and Henkart, P.A. (1996) Different interleukin-1UPbeta converting enzyme (ICE) family protease requirements for the apoptotic death of T lymphocytes triggered by diverse stimuli. J. Exp. Med. 184, 2445–2450.

    Article  CAS  PubMed  Google Scholar 

  14. Armstrong, R.C. et al. (1996) Fas-induced activation of the cell death-related protease CPP32 is inhibited by Bcl-2 and by ICE family protease inhibitors. J. Biol. Chem. 271, 16850–16855.

    Article  CAS  PubMed  Google Scholar 

  15. Garcia-Calvo, M., Peterson, E.P., Leiting, B., Ruel, R., Nicholson, D.W., and Thornberry, N.A. (1998) Inhibition of human caspases by peptide-based macromolecular inhibitors. J. Biol. Chem. 273, 32608–32613.

    Article  CAS  PubMed  Google Scholar 

  16. Sun, X.M., MacFarlane, M., Zhuang, J., Wolf, B.B., Green, D.R., and Cohen, G.M. (1999) Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis. J. Biol. Chem. 274, 5053–5060.

    Article  CAS  PubMed  Google Scholar 

  17. Ekert, P.G., Silke, J., and Vaux, D.L. (1999) Caspase inhibitors. Cell Death Differ. 6, 1081–1086.

    Article  CAS  PubMed  Google Scholar 

  18. Clark, P., Dziarmaga, A., Eccles, M., and Goodyer, P. (2004) Rescue of defective branching nephrogenesis in renal coloboma syndrome by the caspase inhibitor, z-VAD-FMK. J. Am. Soc. Nephrol. 15, 299–305.

    Article  CAS  PubMed  Google Scholar 

  19. Nakano, M. et al. (2004) Caspase-3 inhibitor prevents apoptosis of human islets immediately after isolation and improves islet graft function. Pancreas 29, 104–109.

    Article  CAS  PubMed  Google Scholar 

  20. Nedev, H.N., Klaiman, G., LeBlanc, A., and Saragovi, H.U. (2005) Synthesis and evaluation of novel dipeptidyl benzoyloxymethyl ketones as caspase inhibitors. Biochem. Biophys. Res. Commun. 336, 397–400.

    Article  CAS  PubMed  Google Scholar 

  21. Rauber, P., Angliker, H., Walker, B., and Shaw, E. (1986) The synthesis of peptidylfluoromethanes and their properties as inhibitors of serine proteases and cysteine proteases. Biochem. J. 239, 633–640.

    CAS  PubMed  Google Scholar 

  22. Powers, J.C., Asgian, J.L., Ekici, O.D., and James, K.E. (2002) Irreversible inhibitors of serine, cysteine, and threonine proteases. Chem. Rev. 102, 4639–4750.

    Article  CAS  PubMed  Google Scholar 

  23. Wu, J.C. and Fritz, L.C. (1999) Irreversible caspase inhibitors: tools for studying apoptosis. Methods 17, 320–328.

    Article  CAS  PubMed  Google Scholar 

  24. 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. 259, 308–313.

    Article  CAS  PubMed  Google Scholar 

  25. Amstad, P.A., Yu, G.L., Johnson, G.L., Lee, B.L., Dhawan, S., and Phelps, D.J. (2001) Detection of caspase activation in situ by fluorochrome-labeled caspase inhibitors. Biotechniques 31, 608–616.

    CAS  PubMed  Google Scholar 

  26. Smolewski, P., Bedner, E., Du, L., Hsieh, T.C., Wu, J.M., Phelps, D.J., and Darzynkiewicz, Z. (2001) Detection of caspase activation by fluorochrome-labeled inhibitors: multiparameter analysis by laser scanning cytometry. Cytometry 44, 73–82.

    Article  CAS  PubMed  Google Scholar 

  27. Smolewski, P., Grabarek, J., Halicka, H.D., and Darzynkiewicz, Z. (2002) Assay of caspase activation in situ combined with probing plasma membrane integrity to detect three distinct stages of apoptosis. J. Immunol. Methods 265, 111–121.

    Article  CAS  PubMed  Google Scholar 

  28. 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-a or camptothecin assayed by the stathmo-apoptosis method. Cytometry 47, 143–149.

    Article  CAS  PubMed  Google Scholar 

  29. Wolbers, F., Buijtenhuijs, P., Haanen, C., and Vermes, I. (2004) Apoptotic cell death kinetics in vitro depend on the cell types and the inducers used. Apoptosis 9, 385–392.

    Article  CAS  PubMed  Google Scholar 

  30. Grunewald, S., Paasch, U., Said, T.M., Sharma, R.K., Glander, H.J., and Agarwal, A. (2004) Caspase activation in human spermatozoa in response to physiological and pathological stimuli. Fertil. Steril. 83, 1106–1112.

    Article  Google Scholar 

  31. Fiala, M., Lin, J., Ringman, J., Arab, V.K., Tsao, A., Patel, A., Lossinsky, A.S., Graves, M.C., Gustavson, A., Sayre, J., Sofroni, E., Suarez, T., Chiappelli, F., and Bernard, G. (2005) Ineffective phagocytosis of amyloid-B by macrophages of Alzheimer’s disease patients. J. Alzheimer Dis. 7, 221–232.

    CAS  Google Scholar 

  32. Mizukami, S., Kikuchi, K., Higuchi, T., Urano, Y., Mashima, T., Tsuruo, T., and Nagano, T. (1999) Imaging of caspase-3 activation in HeLa cells stimulated with etoposide using a novel fluorescent probe. FEBS Lett. 453, 356–360.

    Article  CAS  PubMed  Google Scholar 

  33. 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.

    Article  CAS  PubMed  Google Scholar 

  34. Mack, A., Furmann, C., and Hacker, G. (2000) Detection of caspase-activation in intact lymphoid cells using standard caspase substrates and inhibitors. J. Immunol. Methods 241, 19–31.

    Article  CAS  PubMed  Google Scholar 

  35. 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 fluorogenic substrates. J. Exp. Med. 191, 1819–1828.

    Article  CAS  PubMed  Google Scholar 

  36. 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.

    Article  CAS  PubMed  Google Scholar 

  37. Lee, B.W., Johnson, G.L., Hed, S.A., Darzynkiewicz, Z., Talhouk, J.W., and Mehrotra, S. (2003) DEVDase detection in intact apoptotic cells using the cell permeant fluorogenic substrate, (z-DEVD)2-cresyl violet. Biotechniques 35, 1080–1085.

    CAS  PubMed  Google Scholar 

  38. 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.

    Article  CAS  PubMed  Google Scholar 

  39. Duan, R.W., Garner, D.S., Williams, S.D., Funckes-Shippy, C.L., Spath, I.S., and Blomme, E.A. (2003) Comparison of immunohistochemistry for activated caspase-3 and cleaved cytokeratin 18 with TUNEL method for quantification of apoptosis in histological sections of PC-3 subcutaneous xenografts. J. Pathol. 199, 221–228.

    Article  CAS  PubMed  Google Scholar 

  40. Salvioli, S., Ardizzoni, A., Franceschi, C., and Cossarizza, A. (1997) JC-1 but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS Lett. 411, 77–82.

    Article  CAS  PubMed  Google Scholar 

  41. Poot, M., Zhang, Y.Z., Kramer, J.A., Wells, K.S., Jones, L.J., Hanzel, D.K., Lugade, A.G., Singer, V.L., and Haugland, R.P. (1996) Analysis of mitochondrial morphology and function with novel fixable fluorescent stains. J. Histochem. Cytochem. 44, 1363–1372.

    CAS  PubMed  Google Scholar 

  42. Brunner, K.T., Mauel, J., Cerottini, J.C., and Chapuis, B. (1968) Quantitative assay of the lytic action of immune lymphoid cells on 51-Cr-labeled allogenic target cells in vitro; inhibition by isoantibody and by drugs. Immunology 14, 181–196.

    CAS  PubMed  Google Scholar 

  43. Ward, P.W., Bonaparte, M.I., and Barker, E. (2004) HLA-C and HLA-E reduce antibody-dependent natural killer cell-mediated cytotoxicity of HIV-infected primary T cell blasts. AIDS 18, 1769–1779.

    Article  PubMed  Google Scholar 

  44. Barber, D.L., Wherry, E.J., and Ahmed, R. (2003) Cutting edge: rapid in vivo killing by memory CD8 T cells . J. Immunol. 171, 27–31.

    CAS  PubMed  Google Scholar 

  45. Yang, S. and Haluska, F.G. (2004) Treatment of melanoma with 5-fluorouracil or dacarbazine in vitro sensitizes cells to antigen-specific CTL lysis through perforin/granzyme- and Fas-mediated pathways. J. Immunol. 172, 4599–4608.

    CAS  PubMed  Google Scholar 

  46. Schwarer, A.P., Jiang, Y.Z., Deacock, S., Brookes, P.A., Barret, A.J., Goldman, J.M., Batchlor, J.R., and Lechler, R.I. (1994) Comparison of helper and cytotoxic antirecipient T cell frequencies in unrelated bone marrow transplantation. Transplantation 58, 1198–1203.

    CAS  PubMed  Google Scholar 

  47. Russell, J.H. and Ley, T.J. (2002) Lymphocyte-mediated cytotoxicity. Annu. Rev. Immunol. 20, 323–370.

    Article  CAS  PubMed  Google Scholar 

  48. Pross, H., Callewaert, D., and Rubin, P. (1986) Assays for NK cell cytotoxicity-their values and pitfalls, in Immunobiology of Natural Killer Cells (E. Lotzova and R.B. Herberman eds.) Vol. I. CRC Press, Boca Raton, FL, pp. 1–16.

    Google Scholar 

  49. Jerome, K.R., Sloan, D.D., and Aubert, M. (2003) Measurement of CTL-induced cytotoxicity: the caspase 3 assay. Apoptosis 8, 563–571.

    Article  CAS  PubMed  Google Scholar 

  50. Slezak, S.E. and Horan, P.K. (1989) Cell-mediated cytotoxicity: a highly sensitive and informative flow cytometric assay. J. Immunol. Methods 117, 205–214.

    Article  CAS  PubMed  Google Scholar 

  51. Radosevic, K., Garritsen, H.S.P., Van Graft, M., De Grooth, B.G., and Greve, J. (1990) A simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells. J. Immunol. Methods 135, 81–89.

    Article  CAS  PubMed  Google Scholar 

  52. Hatam, L., Schuval, S., and Bonagura, V.R. (1994) Flow cytometric analysis of natural killer cell function as a clinical assay. Cytometry 16, 59–68.

    Article  CAS  PubMed  Google Scholar 

  53. Lee-MacAry, A.E., Ross, E.L., Davies, D., Laylor, R., Honeychurch, J., Glennie, M.J., Snary, D., and Wilkinson, R.W. (2001) Development of a novel flow cytometric cell-mediated cytotoxicity assay using the fluorophores PKH-26 and TO-PRO-3 iodide. J. Immunol. Methods 252, 83–92.

    Article  CAS  PubMed  Google Scholar 

  54. Hoppner, M., Luhm, J., Schlenke, P., Koritke, P., and Frohn, C. (2002) A flow-cytometry based cytotoxicity assay using stained effector cells in combination with native target cells. J. Immunol. Methods 267, 157–163.

    Article  CAS  PubMed  Google Scholar 

  55. Olin, M.R., Choi, K.H., Lee, J., and Molitor, T.W. (2005) γδ T-lymphocyte cytotoxic activity against Mycobacterium bovis analyzed by flow cytometry. J. Immunol. Methods 297, 1–11.

    Article  CAS  PubMed  Google Scholar 

  56. Kienzle, N., Oliver, S., Buttigieg, K., and Kelso, A. (2002) The fluorolysis assay, a highly sensitive method for measuring the cytolytic activity of T cells at very low numbers. J. Immunol. Methods 267, 98–108.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Immunochemistry Technologies, LLC, Bloomington, MN

    Brian W. Lee & Gary L. Johnson

  2. University of Minnesota College of Veterinary Medicine, St. Paul, MN

    Michael R. Olin

  3. University of Minnesota Medical School, Minneapolis, MN

    Robert J. Griffin

Authors
  1. Brian W. Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michael R. Olin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gary L. Johnson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Robert J. Griffin
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Department of Obstetrics Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, CT

    Gil Mor  & Ayesha B. Alvero  & 

Rights and permissions

Reprints and permissions

Copyright information

© 2008 Humana Press Inc.

About this protocol

Cite this protocol

Lee, B.W., Olin, M.R., Johnson, G.L., Griffin, R.J. (2008). In Vitro and In Vivo Apoptosis Detection Using Membrane Permeant Fluorescent-Labeled Inhibitors of Caspases. In: Mor, G., Alvero, A.B. (eds) Apoptosis and Cancer. Methods in Molecular Biology™, vol 414. Humana Press. https://doi.org/10.1007/978-1-59745-339-4_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-339-4_10

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-457-9

  • Online ISBN: 978-1-59745-339-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation