Determining the Extent of Toxicant-Induced Apoptosis Using Concurrent Phased Apoptosis Assays

Part of the Methods in Pharmacology and Toxicology book series (MIPT)


Apoptosis is a stage-dependent process exhibiting characteristic biochemical, molecular and morphological features that vary progressively through the apoptosis process. Apoptosis induced by toxicants may activate varied features of apoptosis to different extents and kinetics. Some of the features activated may occur transiently, while others may not occur in a cell system undergoing toxicant-induced apoptosis. Thus, the best approach for quantitating the extent of toxicant-induced apoptosis involves the utilization of a combination of assays focusing on different morphological, biochemical and molecular features of apoptosis. The kinetics of the apoptosis process will also need to be studied in any cell system subjected to toxicant-induced apoptosis for the first time, especially when more than one compound (such as an apoptosis inducing agent and an inhibitor) is being utilized. The use of multiple concurrent and phased apoptosis assays will streamline the process of determining the extent of apoptosis in such systems. Thus, in this report, we describe the concurrent use of an early, intermediate, and late apoptosis assay in order to measure different biochemical and morphological properties of apoptosis in the same system. We demonstrate the usefulness of the concurrent phased apoptosis assays using human TK6 lymphoblasts undergoing diepoxbutane-induced apoptosis. Additional notes, as well as tips for modifying the protocol for adherent cells, are included.

Key words

Apoptosis Apoptosis assay Concurrent phased apoptosis assays Lymphoblasts Caspase-Glo 3/7 assay pSIVA-IANBD apoptosis assay Nuclear morphology fluorescent dual dye staining assay 


  1. 1.
    Li K, Wu D, Chen X, Zhang T, Zhang L, Yi Y, Miao Z, Jin N, Bi X, Wang H, Xu J, Wang D (2014) Current and emerging biomarkers of cell death in human disease. Biomed Res Int 2014:690103PubMedPubMedCentralGoogle Scholar
  2. 2.
    Goldar S, Khaniani MS, Derakhshan SM, Baradaran B (2015) Molecular mechanisms of apoptosis and roles in cancer development and treatment. Asian Pac J Cancer Prev 16:2129–2144CrossRefPubMedGoogle Scholar
  3. 3.
    Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35:495–516CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Tower J (2015) Programmed cell death in aging. Ageing Res Rev 23:90–100CrossRefPubMedGoogle Scholar
  5. 5.
    Zeng W, Wang X, Xu P, Liu G, Eden HS, Chen X (2015) Molecular imaging of apoptosis: from micro to macro. Theranostics 5:559–582CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Galluzzi L, Vitale I, Abrams JM, Alnemri ES, Baehrecke EH, Blagosklonny MV, Dawson TM, Dawson VL, El-Deiry WS, Fulda S, Gottlieb E, Green DR, Hengartner MO, Kepp O, Knight RA, Kumar S, Lipton SA, Lu X, Madeo F, Malorni W, Mehlen P, Nunez G, Peter ME, Piacentini M, Rubinsztein DC, Shi Y, Simon HU, Vandenabeele P, White E, Yuan J, Zhivotovsky B, Melino G, Kroemer G (2012) Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012. Cell Death Differ 19:107–120CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Galluzzi L, Bravo-San Pedro JM, Vitale I, Aaronson SA, Abrams JM, Adam D, Alnemri ES, Altucci L, Andrews D, Annicchiarico-Petruzzelli M, Baehrecke EH, Bazan NG, Bertrand MJ, Bianchi K, Blagosklonny MV, Blomgren K, Borner C, Bredesen DE, Brenner C, Campanella M, Candi E, Cecconi F, Chan FK, Chandel NS, Cheng EH, Chipuk JE, Cidlowski JA, Ciechanover A, Dawson TM, Dawson VL, De Laurenzi V, De Maria R, Debatin KM, Di Daniele N, Dixit VM, Dynlacht BD, El-Deiry WS, Fimia GM, Flavell RA, Fulda S, Garrido C, Gougeon ML, Green DR, Gronemeyer H, Hajnoczky G, Hardwick JM, Hengartner MO, Ichijo H, Joseph B, Jost PJ et al (2015) Essential versus accessory aspects of cell death: recommendations of the NCCD 2015. Cell Death Differ 22:58–73CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Ulukaya E, Acilan C, Yilmaz Y (2011) Apoptosis: why and how does it occur in biology? Cell Biochem Funct 29:468–480CrossRefPubMedGoogle Scholar
  9. 9.
    Fuchs Y, Steller H (2011) Programmed cell death in animal development and disease. Cell 147:742–758CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Shalini S, Dorstyn L, Dawar S, Kumar S (2015) Old, new and emerging functions of caspases. Cell Death Differ 22:526–539CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Kanemura S, Tsuchiya A, Kanno T, Nakano T, Nishizaki T (2015) Phosphatidylinositol induces caspase-independent apoptosis of malignant pleural mesothelioma cells by accumulating AIF in the nucleus. Cell Physiol Biochem 36:1037–1048CrossRefPubMedGoogle Scholar
  12. 12.
    Rello-Varona S, Herrero-Martin D, Lopez-Alemany R, Munoz-Pinedo C, Tirado OM (2015) “(Not) all (dead) things share the same breath”: identification of cell death mechanisms in anticancer therapy. Cancer Res 75:913–917CrossRefPubMedGoogle Scholar
  13. 13.
    Kim YE, Chen J, Chan JR, Langen R (2010) Engineering a polarity-sensitive biosensor for time-lapse imaging of apoptotic processes and degeneration. Nat Methods 7:67–73CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Kim YE, Chen J, Langen R, Chan JR (2010) Monitoring apoptosis and neuronal degeneration by real-time detection of phosphatidylserine externalization using a polarity-sensitive indicator of viability and apoptosis. Nat Protoc 5:1396–1405CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Bucur O, Stancu AL, Khosravi-Far R, Almasan A (2012) Analysis of apoptosis methods recently used in cancer research and cell death & disease publications. Cell Death Dis 3, e263CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Hankins HM, Baldridge RD, Xu P, Graham TR (2015) Role of flippases, scramblases and transfer proteins in phosphatidylserine subcellular distribution. Traffic 16:35–47CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Leventis PA, Grinstein S (2010) The distribution and function of phosphatidylserine in cellular membranes. Annu Rev Biophys 39:407–427CrossRefPubMedGoogle Scholar
  18. 18.
    Schlegel RA, Williamson P (2001) Phosphatidylserine, a death knell. Cell Death Differ 8:551–563CrossRefPubMedGoogle Scholar
  19. 19.
    Wu H, Che X, Zheng Q, Wu A, Pan K, Shao A, Wu Q, Zhang J, Hong Y (2014) Caspases: a molecular switch node in the crosstalk between autophagy and apoptosis. Int J Biol Sci 10:1072–1083CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Nicholls SB, Hyman BT (2014) Measuring caspase activity in vivo. Methods Enzymol 544:251–269CrossRefPubMedGoogle Scholar
  21. 21.
    McStay GP, Green DR (2014) Measuring apoptosis: caspase inhibitors and activity assays. Cold Spring Harb Protoc 2014:799–806PubMedGoogle Scholar
  22. 22.
    Novus-Biologicals (2014) pSIVA-IANBD Apoptosis/Viability Microscopy Set Product Information and ManualGoogle Scholar
  23. 23.
    Promega (2015) Caspase-Glo 3/7 Technical Bulletin.Google Scholar
  24. 24.
    Squier MK, Cohen JJ (2001) Standard quantitative assays for apoptosis. Mol Biotechnol 19:305–312CrossRefPubMedGoogle Scholar
  25. 25.
    Yadavilli S, Muganda PM (2004) Diepoxybutane induces caspase and p53-mediated apoptosis in human lymphoblasts. Toxicol Appl Pharmacol 195:154–165CrossRefPubMedGoogle Scholar
  26. 26.
    Yadavilli S, Chen Z, Albrecht T, Muganda PM (2009) Mechanism of diepoxybutane-induced p53 regulation in human cells. J Biochem Mol Toxicol 23:373–386CrossRefPubMedGoogle Scholar
  27. 27.
    Jackson MA, Stack HF, Rice JM, Waters MD (2000) A review of the genetic and related effects of 1,3-butadiene in rodents and humans. Mutat Res 463:181–213CrossRefPubMedGoogle Scholar
  28. 28.
    Yadavilli S, Martinez-Ceballos E, Snowden-Aikens J, Hurst A, Joseph T, Albrecht T, Muganda PM (2007) Diepoxybutane activates the mitochondrial apoptotic pathway and mediates apoptosis in human lymphoblasts through oxidative stress. Toxicol In Vitro 21:1429–1441CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Energy and Environmental SystemsNorth Carolina A&T State UniversityGreensboroUSA
  2. 2.Department of BiologyNorth Carolina A&T State UniversityGreensboroUSA

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