Measurement of Cell Death in Mammalian Cells

Brian S. Cummings1, Lauren P. Wills2, Rick G. Schnellmann2

1 Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, Georgia, 2 Department of Pharmaceutical and Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 12.8
DOI:  10.1002/0471141755.ph1208s56
Online Posting Date:  March, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Methods for assessing mammalian cell death are presented in this unit. The unit is divided into six sections: (1) a brief overview of cytotoxicity and pathways of cell death, (2) a method to measure cell death using lactate dehydrogenase (LDH) release as a marker of membrane integrity, (3) a flow cytometry method that simultaneously measures two types of cell death, necrosis, and apoptosis, (4) use of fluorescence microscopy and nuclear morphology to assess apoptosis and necrosis, (5) the use of multi‐well plates and high‐content analysis imaging systems to assess nuclear morphology, and (6) a discussion of the use of cytotoxicity assays to determine the mechanisms of cell death. Curr. Protoc. Pharmacol. 56:12.8.1‐12.8.24. © 2012 by John Wiley & Sons, Inc.

Keywords: apoptosis; necrosis; cytotoxicity

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of Plasma Membrane Integrity and Viability Using LDH Release
  • Basic Protocol 2: Measurement of Necrosis and Apoptosis Using Flow Cytometry
  • Basic Protocol 3: Determination of Nuclear Morphology and Membrane Integrity
  • Alternate Protocol 1: Assessment of Nuclear Morphology and Membrane Integrity Using DAPI and PI
  • Alternate Protocol 2: Assessment of Nuclear Morphology Using Multi‐Well Plates
  • Basic Protocol 4: Measurement of Time‐Dependent Toxicity Using Cell Death Markers
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Measurement of Plasma Membrane Integrity and Viability Using LDH Release

  Materials
  • Cellular material (∼1 mg protein/ml)
  • Toxicants
  • 2:1 (v/v) n‐butyl phthalate/dioctyl phthalate (store at room temperature for up to 1 month)
  • Cell medium or buffer
  • Liquid nitrogen
  • 16 mM pyruvic acid in LDH‐PO 4 buffer (prepare fresh daily)
  • LDH‐PO 4 buffer: 50 mM K 2HPO 4 and 9 mM KH 2PO 4, pH 7.4 (prepare fresh weekly)
  • 0.3 mM β‐NADH in LDH‐PO 4 buffer (prepare fresh daily)
  • 1.5‐ml microcentrifuge tubes
  • 48‐well transparent plate
  • Fluorometric plate reader with a 360‐nm excitation filter (40‐nm bandwidth) and a 460‐nm emission filter (BMG Laboratories, Molecular Devices, and Bio‐Tek)

Basic Protocol 2: Measurement of Necrosis and Apoptosis Using Flow Cytometry

  Materials
  • Cells
  • Toxicant (see Critical Parameters)
  • Phosphate‐buffered saline (PBS; Fisher or Sigma)
  • Annexin binding buffer (see recipe)
  • Annexin V–conjugated FITC (annexin V–FITC), typically at 25 µg/ml (i.e., Biovision or R&D Systems)
  • 1 mg/ml propidium iodide (PI) in PBS
  • Orbital shaker
  • Rubber policeman
  • 50‐µm diameter nylon filter sieves
  • 5‐ml snap‐cap polypropylene tubes
  • Flow cytometer with FL‐1 (FITC) and Fl‐2 (PI) channels
NOTE: The amount of annexin V‐FITC and PI needed for flow cytometry varies among cell types. In addition, the amount of FITC conjugated to annexin V can vary from source to source, and lot to lot. Thus, the amount of annexin V‐FITC and PI used to obtain clear and discrete signals may change two‐ to five‐fold for each cell type. Therefore, pilot experiments must be conducted for optimization.

Basic Protocol 3: Determination of Nuclear Morphology and Membrane Integrity

  Materials
  • Cultured cells on coverslips, in 35‐mm cell culture dishes, or in 96‐well tissue culture plates; or a suspension of cells at a concentration of ∼1 mg protein/ml or 1–2 × 106 cells/ml
  • Phosphate‐buffered saline (PBS)
  • 10% neutral buffered formalin solution (Sigma)
  • 100 µM DAPI in PBS
  • Mounting medium
  • Crystal mount
  • 35‐mm culture dishes
  • Orbital shaker
  • Coverslips
  • Glass slides
  • Fluorometric microscope with 350‐nm excitation and 486‐nm emission filters
  • Centrifuge

Alternate Protocol 1: Assessment of Nuclear Morphology and Membrane Integrity Using DAPI and PI

  • High Content Analysis (HCA) imaging system (e.g., IN Cell Analyzer, GE Healthcare)
  • HCA software package (e.g., Molecular Devices, BD Biosciences, GE Healthcare)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Barry, M., Heibein, J., Pinkoski, M., and Bleackley, R.C. 2000. Quantitative measurement of apoptosis induced by cytotoxic T lymphocytes. Methods Enzymol. 322:40‐46.
   Bortner, C.D. and Cidlowski, J.A. 1999. Caspase independent/dependent regulation of K(+), cell shrinkage, and mitochondrial membrane potential during lymphocyte apoptosis. J. Biol. Chem. 274:21953‐21962.
   Bortner, C.D. and Cidlowski, J.A. 2001. Flow cytometric analysis of cell shrinkage and monovalent ions during apoptosis. Methods Cell Biol. 66:49‐67.
   Brambilla, G., Cavanna, M., Carlo, P., Finollo, R., Sciaba, L., Parodi, S., and Bolognesi, C. 1979. DNA damage and repair induced by diazoacetyl derivatives of amino acids with different mechanism of cytotoxicity. Correlations with mutagenicity and carcinogenicity. J. Cancer Res. Clin. Oncol. 94:7‐20.
  Cande, C., Cohen, I., Daugas, E., Ravagnan, L., Larochette, N., Zamzami, N., and Kroemer, G. 2002. Apoptosis‐inducing factor (AIF): A novel caspase‐independent death effector released from mitochondria. Biochimie 84:215‐222.
   Cummings, B.S. and Schnellmann, R.G. 2001. Pathophysiology of nephrotoxic cell injury. In Diseases of the Kidney and Urinary Tract (R.W. Schrier, ed.) pp. 962‐985. Lippincott Williams & Wilkins, Philadelphia.
   Cummings, B.S. and Schnellmann, R.G. 2002. Cisplatin‐induced renal cell apoptosis: Caspase 3‐dependent and ‐independent pathways. J. Pharmacol. Exp. Ther. 302:8‐17.
   Fanning, J., Biddle, W.C., Goldrosen, M., Crickard, K., Crickard, U., Piver, M.S., and Foon, K.A. 1990. Comparison of cisplatin and carboplatin cytotoxicity in human ovarian cancer cell lines using the MTT assay. Gynecol. Oncol. 39:119‐122.
   Ferlini, C., Biselli, R., Scambia, G., and Fattorossi, A. 1996. Probing chromatin structure in the early phases of apoptosis. Cell Prolif. 29:427‐436.
   Groves, C.E. and Schnellmann, R.G. 1996. Suspensions of rabbit renal proximal tubular cells. In Methods in Renal Toxicology (R.K. Zalups and L.H. Lash, eds.) pp. 147‐162. CRC Press, Boca Raton, Floa.
   Hamel, W., Dazin, P., and Israel, M.A. 1996. Adaptation of a simple flow cytometric assay to identify different stages during apoptosis. Cytometry 25:173‐181.
   Jaeschke, H., Smith, C.W., Clemens, M.G., Ganey, P.E., and Roth, R.A. 1996. Mechanisms of inflammatory liver injury: Adhesion molecules and cytotoxicity of neutrophils. Toxicol. Appl. Pharmacol. 139:213‐226.
   Kerr, J.F. 2002. History of the events leading to the formulation of the apoptosis concept. Toxicology 181‐182:471‐474.
   Lash, L.H. and Tokarz, J.J. 1989. Isolation of two distinct populations of cells from rat kidney cortex and their use in the study of chemical‐induced toxicity. Anal. Biochem. 182:271‐279.
   Lemasters, J.J. 1999. V. Necrapoptosis and the mitochondrial permeability transition: Shared pathways to necrosis and apoptosis. Am. J. Physiol. 276:G1‐6.
   Lemasters, J.J., DiGuiseppi, J., Nieminen, A.L., and Herman, B. 1987. Blebbing, free Ca2+ and mitochondrial membrane potential preceding cell death in hepatocytes. Nature 325:78‐81.
   Lemasters, J.J., Qian, T., Bradham, C.A., Brenner, D.A., Cascio, W.E., Trost, L.C., Nishimura, Y., Nieminen, A.L., and Herman, B. 1999. Mitochondrial dysfunction in the pathogenesis of necrotic and apoptotic cell death. J. Bioenerg. Biomembr. 31:305‐319.
   Lemasters, J.J., Theruvath, T. P., Zhong, Z., and Nieminen, A. 2009. Mitochondrial calcium and the permeability transition in cell death. Biochim. Biophys. Acta 1787:1395‐1401.
   Levin, S., Bucci, T.J., Cohen, S.M., Fix, A.S., Hardisty, J.F., LeGrand, E.K., Maronpot, R.R., and Trump, B.F. 1999. The nomenclature of cell death: Recommendations of an ad hoc Committee of the Society of Toxicologic Pathologists. Toxicol. Pathol. 27:484‐490.
   Licht, R., Jacobs, C.W., Tax, W.J., and Berden, J.H. 1999. An assay for the quantitative measurement of in vitro phagocytosis of early apoptotic thymocytes by murine resident peritoneal macrophages. J. Immunol. Methods 223:237‐248.
   Lieberthal, W., Triaca, V., and Levine, J. 1996. Mechanisms of death induced by cisplatin in proximal tubular epithelial cells: Apoptosis vs. necrosis. Am. J. Physiol. 270:F700‐F708.
   Lindsten, T.A., Ross, J., King, A., Zong, W.X., Rathmell, J.C., Shiels, H.A., Ulrich, E., Waymire, K.G., Mahar, P., Frauwirth, K., Chen, Y., Wei, M., Eng, V.M., Adelman, D.M., Simon, M.C., Ma, A., Golden, J.A., Evan, G., Korsmeyer, S.J., MacGregor, G.R., and Thompson, C.B. 2000. The combined functions of proapoptotic Bcl‐2 family members bak and bax are essential from normal development of multiple tissues. Mol. Cell. 6:1389‐1399.
   Lipton, S.A. and Nicotera, P. 1998. Calcium, free radicals and excitotoxins in neuronal apoptosis. Cell Calcium 23:165‐171.
   Liu, X., Kim, C., Yang, J., Jemmerson, R., and Wang, X. 1996. Induction of apoptotic program in cell‐free extracts: Requirement for dATP and cytochrome c. Cell 86:147‐157.
   Liu, X., Harriman, J.F., and Schnellmann, R.G. 2002. Cytoprotective properties of novel nonpeptide calpain inhibitors in renal cells. J. Pharmacol. Exp. Ther. 302:88‐94.
   Majno, G. and Joris, I. 1995. Apoptosis, necrosis, and necrosis. An overview of cell death. Am. J. Pathol. 146:3‐15.
   Mertens, J.J., Gibson, N.W., Lau, S.S., and Monks, T.J. 1995. Reactive oxygen species and DNA damage in 2‐bromo‐(glutathion‐S‐yl) hydroquinone‐mediated cytotoxicity. Arch. Biochem. Biophys. 320:51‐58.
   Modha, K., Whiteside, J.P., and Spier, R.E. 1993. The determination of cellular viability of hybridoma cells in microtitre plates: A colorimetric assay based on neutral red. Cytotechnology 13:227‐232.
   Monks, T.J., Highet, R.J., and Lau, S.S. 1988. 2‐Bromo‐(diglutathion‐S‐yl)hydroquinone nephrotoxicity: Physiological, biochemical, and electrochemical determinants. Mol. Pharmacol. 34:492‐500.
   Moran, J.H. and Schnellmann, R.G. 1996. A rapid beta‐NADH‐linked fluorescence assay for lactate dehydrogenase in cellular death. J. Pharmacol. Toxicol. Methods 36:41‐44.
   Nony, P.A. and Schnellmann, R.G. 2001. Interactions between collagen IV and collagen‐binding integrins in renal cell repair after sublethal injury. Mol. Pharmacol. 60:1226‐1234.
   Norberg, E., Orrenius, S., and Zhivotovsky, B. 2010. Mitochondrial regulation of cell death: Processing of apoptosis‐inducing factor (AIF). Biochem. Biophys. Res. Commun. 396:95‐100.
   Ogden, D., Khodakhah, K., Carter, T., Thomas, M., and Capiod, T. 1995. Analogue computation of transient changes of intracellular free Ca2+ concentration with the low affinity Ca2+ indicator furaptra during whole‐cell patch‐clamp recording. Pflugers Arch. 429:587‐591.
   Orrenius, S., Nicotera, P., and Zhivotovsky, B. 2011. Cell death mechanisms and their implications in toxicology. Tox. Sci. 119:3‐19.
   Otsuki, Y., Li, Z., and Shibata, M.A. 2003. Apoptotic detection methods‐from morphology to gene. Prog. Histochem. Cytochem. 38:275‐339.
   Proskuryakov, S.Y. and Gabai, V.L. 2010. Mechanisms of tumor cell necrosis. Curr. Pharm. Des. 16:56‐68.
   Reers, M., Smiley, S.T., Mottola‐Hartshorn, C., Chen, A., Lin, M., and Chen, L.B. 1995. Mitochondrial membrane potential monitored by JC‐1 dye. Methods Enzymol. 260:406‐417.
   Saraste, A. 1999. Morphologic criteria and detection of apoptosis. Herz 24:189‐195.
   Saraste, A. and Pulkki, K. 2000. Morphologic and biochemical hallmarks of apoptosis. Cardiovasc. Res. 45:528‐537.
   Schnellmann, R. 1994. Measurement of O2 consumption. In In Vitro Toxicity Indicators (C. Tyson and J. Frazier, eds.) pp. 128‐139. Academic Press, San Diego.
   Schnellmann, R.G. and Mandel, L.J. 1986. Cellular toxicity of bromobenzene and bromobenzene metabolites to rabbit proximal tubules: The role and mechanism of 2‐bromohydroquinone. J. Pharmacol. Exp. Ther. 237:456‐461.
   Schnellmann, R.G., Cross, T.J., and Lock, E.A. 1989. Pentachlorobutadienyl‐L‐cysteine uncouples oxidative phosphorylation by dissipating the proton gradient. Toxicol. Appl. Pharmacol. 100:498‐505.
   Schutte, B., Nuydens, R., Geerts, H., and Ramaekers, F. 1998. Annexin V binding assay as a tool to measure apoptosis in differentiated neuronal cells. J. Neurosci. Methods 86:63‐69.
   Shen, W., Kamendulis, L.M., Ray, S.D., and Corcoran, G.B. 1991. Acetaminophen‐induced cytotoxicity in cultured mouse hepatocytes: Correlation of nuclear Ca2+ accumulation and early DNA fragmentation with cell death. Toxicol. Appl. Pharmacol. 111:242‐254.
   Singh, N.P. 2000. A simple method for accurate estimation of apoptotic cells. Exp. Cell Res. 256:328‐337.
   Sorger, T. and Germinario, R.J. 1983. A direct solubilization procedure for the determination of DNA and protein in cultured fibroblast monolayers. Anal. Biochem. 131:254‐256.
   Takemura, G., Kato, S., Aoyama, T., Hayakawa, Y., Kanoh, M., Maruyama, R., Arai, M., Nishigaki, K., Minatoguchi, S., Fukuda, K., Fujiwara, T., and Fujiwara, H. 2001. Characterization of ultrastructure and its relation with DNA fragmentation in Fas‐induced apoptosis of cultured cardiac myocytes. J. Pathol. 193:546‐56.
   Tait, J. 2008. Imaging of apoptosis. J. Nucl Med. 49:1573‐1576.
   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., Vaillancourt, J.P., Chapman, K.T., and Nicholson, D.W. 1997. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J. Biol. Chem. 272:17907‐17911.
   Trump, B.F., Berezesky, I.K., Chang, S.H., and Phelps, P.C. 1997. The pathways of cell death: Necrosis, apoptosis, and necrosis. Toxicol. Pathol. 25:82‐88.
   Vanags, D.M., Porn‐Ares, M.I., Coppola, S., Burgess, D.H., and Orrenius, S. 1996. Protease involvement in fodrin cleavage and phosphatidylserine exposure in apoptosis. J. Biol. Chem. 271:31075‐31085.
   Warnes, G. and Martins, S. 2011. Real‐time flow cytometry for the kinetic analysis of oncosis. Cytometry A 79:181‐191.
   Weaver, V.M., Lach, B., Walker, P.R., and Sikorska, M. 1993. Role of proteolysis in apoptosis: Involvement of serine proteases in internucleosomal DNA fragmentation in immature thymocytes. Biochem. Cell Biol. 71:488‐500.
   Yan, X., Habbersett, R.C., Cordek, J.M., Nolan, J.P., Yoshida, T.M, Jett, J.H., and Marrone, B.L. 2000. Development of a mechanism‐based, DNA staining protocol using SYTOX orange nucleic acid stain and DNA fragment sizing flow cytometry. Anal. Biochem. 286:138‐148.
   Yang, H., Acker, J., Chen, A., and McGann, L., 1998. In situ assessment of cell viability. Cell Transplant. 7:443‐4451.
   Zhang, J., Reedy, M.C., Hannun, Y.A., and Obeid, L.M. 1999. Inhibition of caspases inhibits the release of apoptotic bodies: Bcl‐2 inhibits the initiation of formation of apoptotic bodies in chemotherapeutic agent‐induced apoptosis. J. Cell Biol. 145:99‐108.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library