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Methods to Study and Distinguish Necroptosis

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Necrotic Cell Death

Part of the book series: Cell Death in Biology and Diseases ((CELLDEATH))

Abstract

During the decade the importance of regulated necrotic cell death in several pathologies has been increasingly recognized. Intensified research has uncovered many pathways involved in regulated necrosis (RN) that has resulted in several neologisms. The best characterized form of RN is induced by receptor-interacting protein kinase-1 and -3, coined as necroptosis. Unfortunately, until now there are no positive and distinguishing markers available for any of these forms of regulated necrosis, let alone a single marker that identifies regulated necrosis from apoptosis. Here we present an overview of different methods that can be used to discriminate apoptosis from necrosis, more in particular necroptosis, both in vivo and in vitro. Methods for necrotic cell death detection are summarized, and potential pitfalls of these methods are briefly discussed. Basically, a sequential approach is proposed to determine the cell death modality by following a decision tree, allowing discrimination between apoptosis and different forms of RN. Finally, pharmacological and transgenic methods for dissecting subroutines in RN are mentioned.

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References

  • Andersson U, Wang H, Palmblad K, Aveberger AC, Bloom O, Erlandsson-Harris H, Janson A, Kokkola R, Zhang M, Yang H, Tracey KJ (2000) High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes. J Exp Med 192:565–570

    PubMed Central  PubMed  CAS  Google Scholar 

  • Awad D, Schrader I, Bartok M, Mohr A, Gabel D (2011) Comparative toxicology of trypan blue, brilliant blue G, and their combination together with polyethylene glycol on human pigment epithelial cells. Invest Ophthalmol Vis Sci 52:4085–4090

    PubMed  CAS  Google Scholar 

  • Baines C, Kaiser R, Purcell N, Blair N, Osinska H, Hambleton M, Brunskill E, Sayen M, Gottlieb R, Dorn G, Robbins J, Molkentin J (2005) Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature 434:658–662

    PubMed  CAS  Google Scholar 

  • Bartok E, Bauernfeind F, Khaminets MG, Jakobs C, Monks B, Fitzgerald KA, Latz E, Hornung V (2013) iGLuc: a luciferase-based inflammasome and protease activity reporter. Nat Methods 10:147–154

    PubMed  CAS  Google Scholar 

  • Bernhard D, Schwaiger W, Crazzolara R, Tinhofer I, Kofler R, Csordas A (2003) Enhanced MTT-reducing activity under growth inhibition by resveratrol in CEM-C7H2 lymphocytic leukemia cells. Cancer Lett 195:193–199

    PubMed  CAS  Google Scholar 

  • Blankenberg FG, Katsikis PD, Tait JF, Davis RE, Naumovski L, Ohtsuki K, Kopiwoda S, Abrams MJ, Strauss HW (1999) Imaging of apoptosis (programmed cell death) with 99mTc annexin V. J Nucl Med 40:184–191

    PubMed  CAS  Google Scholar 

  • Chiaradia M, Baranes L, Van Nhieu JT, Vignaud A, Laurent A, Decaens T, Charles-Nelson A, Brugières P, Katsahian S, Djabbari M, Deux JF, Sobhani I, Karoui M, Rahmouni A, Luciani A (2014) Intravoxel incoherent motion (IVIM) MR imaging of colorectal liver metastases: Are we only looking at tumor necrosis? J Magn Reson Imaging 38:27-40

    Google Scholar 

  • Cho YS, Challa S, Moquin D, Genga R, Ray TD, Guildford M, Chan FK (2009) Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 137:1112–1123

    PubMed Central  PubMed  CAS  Google Scholar 

  • Christofferson DE, Yuan J (2010) Cyclophilin A release as a biomarker of necrotic cell death. Cell Death Differ 17:1942–1943

    PubMed Central  PubMed  CAS  Google Scholar 

  • Cohen A, Shirvan A, Levin G, Grimberg H, Reshef A, Ziv I (2009) From the Gla domain to a novel small-molecule detector of apoptosis. Cell Res 19:625–637

    PubMed  CAS  Google Scholar 

  • Cohen I, Rider P, Carmi Y, Braiman A, Dotan S, White MR, Voronov E, Martin MU, Dinarello CA, Apte RN (2010) Differential release of chromatin-bound IL-1alpha discriminates between necrotic and apoptotic cell death by the ability to induce sterile inflammation. Proc Natl Acad Sci U S A 107:2574–2579

    PubMed Central  PubMed  CAS  Google Scholar 

  • Cordeiro MF, Guo L, Coxon KM, Duggan J, Nizari S, Normando EM, Sensi SL, Sillito AM, Fitzke FW, Salt TE, Moss SE (2010) Imaging multiple phases of neurodegeneration: a novel approach to assessing cell death in vivo. Cell Death Dis 1:e3

    PubMed Central  PubMed  CAS  Google Scholar 

  • Crawford ED, Wells JA (2011) Caspase substrates and cellular remodeling. Annu Rev Biochem 80:1055–1087

    PubMed  CAS  Google Scholar 

  • Cummings J, Ward TH, Greystoke A, Ranson M, Dive C (2008) Biomarker method validation in anticancer drug development. Br J Pharmacol 153:646–656

    PubMed Central  PubMed  CAS  Google Scholar 

  • Cummings MC, Winterford CM, Walker NI (1997) Apoptosis. Am J Surg Pathol 21:88–101

    PubMed  CAS  Google Scholar 

  • Dasari M, Lee S, Sy J, Kim D, Brown M, Davis M, Murthy N (2010) Hoechst-IR: an imaging agent that detects necrotic tissue in vivo by binding extracellular DNA. Org Lett 12:3300–3303

    PubMed Central  PubMed  CAS  Google Scholar 

  • Dave SR, White CC, Gao X, Kavanagh TJ (2012) Luminescent quantum dots for molecular toxicology. Adv Exp Med Biol 745:117–137

    PubMed  CAS  Google Scholar 

  • De Saint-Hubert M, Prinsen K, Mortelmans L, Verbruggen A, Mottaghy FM (2009) Molecular imaging of cell death. Methods 48:178–187

    PubMed  Google Scholar 

  • De Valck D, Beyaert R, Van Roy F, Fiers W (1993) Tumor necrosis factor cytotoxicity is associated with phospholipase D activation. Eur J Biochem/FEBS 212:491–497

    Google Scholar 

  • Degterev A, Hitomi J, Germscheid M, Ch’en IL, Korkina O, Teng X, Abbott D, Cuny GD, Yuan C, Wagner G, Hedrick SM, Gerber SA, Lugovskoy A, Yuan J (2008) Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol 4:313–321

    PubMed  CAS  Google Scholar 

  • Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, Cuny GD, Mitchison TJ, Moskowitz MA, Yuan J (2005) Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol 1:112–119

    PubMed  CAS  Google Scholar 

  • Degterev A, Maki J, Yuan J (2013) Activity and specificity of necrostatin-1, small-molecule inhibitor of RIP1 kinase. Cell Death Differ 20:366

    PubMed Central  PubMed  CAS  Google Scholar 

  • Delgado ME, Olsson M, Lincoln FA, Zhivotovsky B, Rehm M (2013) Determining the contributions of caspase-2, caspase-8 and effector caspases to intracellular VDVADase activities during apoptosis initiation and execution. Biochim Biophys Acta 1833:2279–2292

    PubMed  CAS  Google Scholar 

  • Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, Patel DN, Bauer AJ, Cantley AM, Yang WS, Morrison B 3rd, Stockwell BR (2012) Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149:1060–1072

    PubMed Central  PubMed  CAS  Google Scholar 

  • Dondelinger Y, Aguileta MA, Goossens V, Dubuisson C, Grootjans S, Dejardin E, Vandenabeele P, Bertrand MJ (2013) RIPK3 contributes to TNFR1-mediated RIPK1 kinase-dependent apoptosis in conditions of cIAP1/2 depletion or TAK1 kinase inhibition. Cell Death Differ 20:1381–1392

    Google Scholar 

  • Duan WR, Garner DS, Williams SD, Funckes-Shippy CL, Spath IS, Blomme EA (2003) Comparison of immunohistochemistry for activated caspase-3 and cleaved cytokeratin 18 with the TUNEL method for quantification of apoptosis in histological sections of PC-3 subcutaneous xenografts. J Pathol 199:221–228

    PubMed  CAS  Google Scholar 

  • Duprez L, Bertrand MJ, Vanden Berghe T, Dondelinger Y, Festjens N, Vandenabeele P (2012) Intermediate domain of receptor-interacting protein kinase 1 (RIPK1) determines switch between necroptosis and RIPK1 kinase-dependent apoptosis. J Biol Chem 287:14863–14872

    PubMed Central  PubMed  CAS  Google Scholar 

  • Duprez L, Takahashi N, Van Hauwermeiren F, Vandendriessche B, Goossens V, Vanden Berghe T, Declercq W, Libert C, Cauwels A, Vandenabeele P (2011) RIP kinase-dependent necrosis drives lethal systemic inflammatory response syndrome. Immunity 35:908–918

    PubMed  CAS  Google Scholar 

  • Festjens N, Kalai M, Smet J, Meeus A, Van Coster R, Saelens X, Vandenabeele P (2006) Butylated hydroxyanisole is more than a reactive oxygen species scavenger. Cell Death Differ 13:166–169

    PubMed  CAS  Google Scholar 

  • Figueroa RA, Ramberg V, Gatsinzi T, Samuelsson M, Zhang M, Iverfeldt K, Hallberg E (2011) Anchored FRET sensors detect local caspase activation prior to neuronal degeneration. Mol Neurodegener 6:35

    PubMed Central  PubMed  CAS  Google Scholar 

  • Fonge H, Chitneni SK, Lixin J, Vunckx K, Prinsen K, Nuyts J, Mortelmans L, Bormans G, Ni Y, Verbruggen A (2007) Necrosis avidity of (99m)Tc(CO)3-labeled pamoic acid derivatives: synthesis and preliminary biological evaluation in animal models of necrosis. Bioconjug Chem 18:1924–1934

    PubMed  CAS  Google Scholar 

  • Galban S, Jeon YH, Bowman BM, Stevenson J, Sebolt KA, Sharkey LM, Lafferty M, Hoff BA, Butler BL, Wigdal SS, Binkowski BF, Otto P, Zimmerman K, Vidugiris G, Encell LP, Fan F, Wood KV, Galban CJ, Ross BD, Rehemtulla A (2013) Imaging proteolytic activity in live cells and animal models. PLoS One 8:e66248

    PubMed Central  PubMed  CAS  Google Scholar 

  • 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 19:107–120

    PubMed Central  PubMed  CAS  Google Scholar 

  • Gavrieli Y, Sherman Y, Ben-Sasson SA (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493–501

    PubMed  CAS  Google Scholar 

  • Goossens V, Grooten J, De Vos K, Fiers W (1995) Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity. Proc Natl Acad Sci U S A 92:8115–8119

    PubMed Central  PubMed  CAS  Google Scholar 

  • Goossens V, Stange G, Moens K, Pipeleers D, Grooten J (1999) Regulation of tumor necrosis factor-induced, mitochondria- and reactive oxygen species-dependent cell death by the electron flux through the electron transport chain complex I. Antioxid Redox Signal 1:285–295

    PubMed  CAS  Google Scholar 

  • Grimberg H, Levin G, Shirvan A, Cohen A, Yogev-Falach M, Reshef A, Ziv I (2009) Monitoring of tumor response to chemotherapy in vivo by a novel small-molecule detector of apoptosis. Apoptosis 14:257–267

    PubMed  CAS  Google Scholar 

  • Gukovskaya AS, Perkins P, Zaninovic V, Sandoval D, Rutherford R, Fitzsimmons T, Pandol SJ, Poucell-Hatton S (1996) Mechanisms of cell death after pancreatic duct obstruction in the opossum and the rat. Gastroenterology 110:875–884

    PubMed  CAS  Google Scholar 

  • Gunther C, Martini E, Wittkopf N, Amann K, Weigmann B, Neumann H, Waldner MJ, Hedrick SM, Tenzer S, Neurath MF, Becker C (2011) Caspase-8 regulates TNF-alpha-induced epithelial necroptosis and terminal ileitis. Nature 477:335–339

    PubMed Central  PubMed  Google Scholar 

  • He L, Wu X, Meylan F, Olson DP, Simone J, Hewgill D, Siegel R, Lipsky PE (2004) Monitoring caspase activity in living cells using fluorescent proteins and flow cytometry. Am J Pathol 164:1901–1913

    PubMed Central  PubMed  CAS  Google Scholar 

  • He S, Wang L, Miao L, Wang T, Du F, Zhao L, Wang X (2009) Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell 137:1100–1111

    PubMed  CAS  Google Scholar 

  • Herbst KJ, Allen MD, Zhang J (2011) Luminescent kinase activity biosensors based on a versatile bimolecular switch. J Am Chem Soc 133:5676–5679

    PubMed Central  PubMed  CAS  Google Scholar 

  • Holler N, Zaru R, Micheau O, Thome M, Attinger A, Valitutti S, Bodmer JL, Schneider P, Seed B, Tschopp J (2000) Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol 1:489–495

    PubMed  CAS  Google Scholar 

  • Hsu PD, Scott DA, Weinstein J, Ran F, Konermann S, Agarwala V, Li Y, Fine E, Wu X, Shalem O, Cradick T, Marraffini L, Bao G, Zhang F (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol 31:827–832

    Google Scholar 

  • Jouan-Lanhouet S, Arshad MI, Piquet-Pellorce C, Martin-Chouly C, Le Moigne-Muller G, Van Herreweghe F, Takahashi N, Sergent O, Lagadic-Gossmann D, Vandenabeele P, Samson M, Dimanche-Boitrel MT (2012) TRAIL induces necroptosis involving RIPK1/RIPK3-dependent PARP-1 activation. Cell Death Differ 19:2003–2014

    PubMed Central  PubMed  CAS  Google Scholar 

  • Kaiser AM, Saluja AK, Sengupta A, Saluja M, Steer ML (1995) Relationship between severity, necrosis, and apoptosis in five models of experimental acute pancreatitis. Am J Physiol 269:C1295–C1304

    PubMed  CAS  Google Scholar 

  • Kang TB, Yang SH, Toth B, Kovalenko A, Wallach D (2013) Caspase-8 Blocks Kinase RIPK3-Mediated Activation of the NLRP3 Inflammasome. Immunity 38:27–40

    Google Scholar 

  • Kelliher MA, Grimm S, Ishida Y, Kuo F, Stanger BZ, Leder P (1998) The death domain kinase RIP mediates the TNF-induced NF-kappaB signal. Immunity 8:297–303

    PubMed  CAS  Google Scholar 

  • Kerr JF, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26:239–257

    PubMed Central  PubMed  CAS  Google Scholar 

  • Kersse K, Verspurten J, Vanden Berghe T, Vandenabeele P (2011) The death-fold superfamily of homotypic interaction motifs. Trends Biochem Sci 36:541–552

    PubMed  CAS  Google Scholar 

  • Kim HJ, Koo SY, Ahn BH, Park O, Park DH, Seo DO, Won JH, Yim HJ, Kwak HS, Park HS, Chung CW, Oh YL, Kim SH (2010) NecroX as a novel class of mitochondrial reactive oxygen species and ONOO(−) scavenger. Arch Pharm Res 33:1813–1823

    PubMed  CAS  Google Scholar 

  • Kim YS, Morgan MJ, Choksi S, Liu ZG (2007) TNF-induced activation of the Nox1 NADPH oxidase and its role in the induction of necrotic cell death. Mol Cell 26:675–687

    PubMed  CAS  Google Scholar 

  • Knott G, Rosset S, Cantoni M (2011) Focussed ion beam milling and scanning electron microscopy of brain tissue. J Vis Exp (53):e2588

    Google Scholar 

  • Kockx MM, Muhring J, Knaapen MW, de Meyer GR (1998) RNA synthesis and splicing interferes with DNA in situ end labeling techniques used to detect apoptosis. Am J Pathol 152:885–888

    PubMed Central  PubMed  CAS  Google Scholar 

  • Kominami K, Nagai T, Sawasaki T, Tsujimura Y, Yashima K, Sunaga Y, Tsuchimochi M, Nishimura J, Chiba K, Nakabayashi J, Koyamada K, Endo Y, Yokota H, Miyawaki A, Manabe N, Sakamaki K (2012) In vivo imaging of hierarchical spatiotemporal activation of caspase-8 during apoptosis. PLoS One 7:e50218

    PubMed Central  PubMed  CAS  Google Scholar 

  • Kourtis N, Nikoletopoulou V, Tavernarakis N (2012) Small heat-shock proteins protect from heat-stroke-associated neurodegeneration. Nature 490:213–218

    PubMed  CAS  Google Scholar 

  • Krysko DV, Agostinis P, Krysko O, Garg AD, Bachert C, Lambrecht BN, Vandenabeele P (2011) Emerging role of damage-associated molecular patterns derived from mitochondria in inflammation. Trends Immunol 32:157–164

    PubMed  CAS  Google Scholar 

  • Krysko DV, Brouckaert G, Kalai M, Vandenabeele P, D’Herde K (2003) Mechanisms of internalization of apoptotic and necrotic L929 cells by a macrophage cell line studied by electron microscopy. J Morphol 258:336–345

    PubMed  Google Scholar 

  • Kuenzler KA, Pearson PY, Schwartz MZ (2002) IL-11 pretreatment reduces cell death after intestinal ischemia-reperfusion. J Surg Res 108:268–272

    PubMed  CAS  Google Scholar 

  • Kuranaga E (2012) Beyond apoptosis: caspase regulatory mechanisms and functions in vivo. Genes Cells 17:83–97

    PubMed  CAS  Google Scholar 

  • Lamkanfi M, Festjens N, Declercq W, Vanden Berghe T, Vandenabeele P (2007) Caspases in cell survival, proliferation and differentiation. Cell Death Differ 14:44–55

    PubMed  CAS  Google Scholar 

  • Laster SM, Wood JG, Gooding LR (1988) Tumor necrosis factor can induce both apoptotic and necrotic forms of cell lysis. J Immunol 141:2629–2634

    PubMed  CAS  Google Scholar 

  • Laussmann MA, Passante E, Dussmann H, Rauen JA, Wurstle ML, Delgado ME, Devocelle M, Prehn JH, Rehm M (2011) Proteasome inhibition can induce an autophagy-dependent apical activation of caspase-8. Cell Death Differ 18:1584–1597

    PubMed Central  PubMed  CAS  Google Scholar 

  • Li J, Cona MM, Chen F, Feng Y, Zhou L, Yu J, Nuyts J, de Witte P, Zhang J, Himmelreich U, Verbruggen A, Ni Y (2012) Exploring theranostic potentials of radioiodinated hypericin in rodent necrosis models. Theranostics 2:1010–1019

    PubMed Central  PubMed  CAS  Google Scholar 

  • Linkermann A, Bräsen JH, Darding M, Jin MK, Sanz AB, Heller JO, De Zen F, Weinlich R, Ortiz A, Walczak H, Weinberg JM, Green DR, Kunzendorf U, Krautwald S (2013) Two independent pathways of regulated necrosis mediate ischemia-reperfusion injury. Proc Natl Acad Sci U S A 110:12024–12029

    Google Scholar 

  • Louis KS, Siegel AC (2011) Cell viability analysis using trypan blue: manual and automated methods. Methods Mol Biol 740:7–12

    PubMed  CAS  Google Scholar 

  • Luthi AU, Cullen SP, McNeela EA, Duriez PJ, Afonina IS, Sheridan C, Brumatti G, Taylor RC, Kersse K, Vandenabeele P, Lavelle EC, Martin SJ (2009) Suppression of interleukin-33 bioactivity through proteolysis by apoptotic caspases. Immunity 31:84–98

    PubMed  CAS  Google Scholar 

  • Mahajan NP, Harrison-Shostak DC, Michaux J, Herman B (1999) Novel mutant green fluorescent protein protease substrates reveal the activation of specific caspases during apoptosis. Chem Biol 6:401–409

    PubMed  CAS  Google Scholar 

  • Mariani G, Villa G, Rossettin PF, Spallarossa P, Bezante GP, Brunelli C, Pak KY, Khaw BA, Strauss HW (1999) Detection of acute myocardial infarction by 99mTc-labeled d-glucaric acid imaging in patients with acute chest pain. J Nucl Med 40:1832–1839

    PubMed  CAS  Google Scholar 

  • Morris MC (2013) Fluorescent biosensors—probing protein kinase function in cancer and drug discovery. Biochim Biophys Acta 1834:1387–1395

    PubMed  CAS  Google Scholar 

  • Nagata S (2000) Apoptotic DNA fragmentation. Exp Cell Res 256:12–18

    PubMed  CAS  Google Scholar 

  • Nagata S (2002) Breakdown of chromosomal DNA. Cornea 21:S2–S6

    PubMed  Google Scholar 

  • Nakagawa T, Shimizu S, Watanabe T, Yamaguchi O, Otsu K, Yamagata H, Inohara H, Kubo T, Tsujimoto Y (2005) Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death. Nature 434:652–658

    PubMed  CAS  Google Scholar 

  • Napirei M, Wulf S, Mannherz HG (2004) Chromatin breakdown during necrosis by serum Dnase1 and the plasminogen system. Arthritis Rheum 50:1873–1883

    PubMed  CAS  Google Scholar 

  • Nguyen QD, Smith G, Glaser M, Perumal M, Arstad E, Aboagye EO (2009) Positron emission tomography imaging of drug-induced tumor apoptosis with a caspase-3/7 specific [18F]-labeled isatin sulfonamide. Proc Natl Acad Sci U S A 106:16375–16380

    PubMed Central  PubMed  CAS  Google Scholar 

  • Ni Y, Huyghe D, Verbeke K, de Witte PA, Nuyts J, Mortelmans L, Chen F, Marchal G, Verbruggen AM, Bormans GM (2006) First preclinical evaluation of mono-[123I]iodohypericin as a necrosis-avid tracer agent. Eur J Nucl Med Mol Imaging 33:595–601

    PubMed  CAS  Google Scholar 

  • Ni Y, Petre C, Miao Y, Yu J, Cresens E, Adriaens P, Bosmans H, Semmler W, Baert AL, Marchal G (1997) Magnetic resonance imaging-histomorphologic correlation studies on paramagnetic metalloporphyrins in rat models of necrosis. Invest Radiol 32:770–779

    PubMed  CAS  Google Scholar 

  • Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139:271–279

    PubMed  CAS  Google Scholar 

  • Perfetto SP, Chattopadhyay PK, Lamoreaux L, Nguyen R, Ambrozak D, Koup RA, Roederer M (2006) Amine reactive dyes: an effective tool to discriminate live and dead cells in polychromatic flow cytometry. J Immunol Methods 313:199–208

    PubMed  CAS  Google Scholar 

  • Pick E, Keisari Y (1980) A simple colorimetric method for the measurement of hydrogen peroxide produced by cells in culture. J Immunol Methods 38:161–170

    PubMed  CAS  Google Scholar 

  • Pop C, Salvesen GS (2009) Human caspases: activation, specificity, and regulation. J Biol Chem 284:21777–21781

    PubMed Central  PubMed  CAS  Google Scholar 

  • Pop C, Salvesen GS, Scott FL (2008) Caspase assays: identifying caspase activity and substrates in vitro and in vivo. Methods Enzymol 446:351–367

    PubMed  CAS  Google Scholar 

  • Prinsen K, Jin L, Vunckx K, De Saint-Hubert M, Zhou L, Cleynhens J, Nuyts J, Bormans G, Ni Y, Verbruggen A (2011) Radiolabeling and preliminary biological evaluation of a (99m)Tc(CO)(3) labeled 3,3′-(benzylidene)-bis-(1H-indole-2-carbohydrazide) derivative as a potential SPECT tracer for in vivo visualization of necrosis. Bioorg Med Chem Lett 21:502–505

    PubMed  CAS  Google Scholar 

  • Rehm M, Dussmann H, Prehn JH (2003) Real-time single cell analysis of Smac/DIABLO release during apoptosis. J Cell Biol 162:1031–1043

    PubMed Central  PubMed  CAS  Google Scholar 

  • Remijsen Q, Goossens V, Grootjans S, Van den Haute C, Vanlangenakker N, Dondelinger Y, Roelandt R, Bruggeman I, Goncalves A, Bertrand MJ, Baekelandt V, Takahashi N, Berghe TV, Vandenabeele P (2014) Depletion of RIPK3 or MLKL blocks TNF-driven necroptosis and switches towards a delayed RIPK1 kinase-dependent apoptosis. Cell Death Dis 5:e1004. doi:10.1038/cddis.2013.531. PMID: 24434512

  • Scaffidi P, Misteli T, Bianchi ME (2002) Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 418:191–195

    PubMed  CAS  Google Scholar 

  • Schulze-Osthoff K, Beyaert R, Vandevoorde V, Haegeman G, Fiers W (1993) Depletion of the mitochondrial electron transport abrogates the cytotoxic and gene-inductive effects of TNF. EMBO J 12:3095–3104

    PubMed Central  PubMed  CAS  Google Scholar 

  • Seiler A, Schneider M, Forster H, Roth S, Wirth EK, Culmsee C, Plesnila N, Kremmer E, Radmark O, Wurst W, Bornkamm GW, Schweizer U, Conrad M (2008) Glutathione peroxidase 4 senses and translates oxidative stress into 12/15-lipoxygenase dependent- and AIF-mediated cell death. Cell Metab 8:237–248

    PubMed  CAS  Google Scholar 

  • Shen S, Kepp O, Michaud M, Martins I, Minoux H, Metivier D, Maiuri MC, Kroemer RT, Kroemer G (2011) Association and dissociation of autophagy, apoptosis and necrosis by systematic chemical study. Oncogene 30:4544–4556

    PubMed  CAS  Google Scholar 

  • Shinzawa K, Tsujimoto Y (2003) PLA2 activity is required for nuclear shrinkage in caspase-independent cell death. J Cell Biol 163:1219–1230

    PubMed Central  PubMed  CAS  Google Scholar 

  • Shu X, Lev-Ram V, Deerinck TJ, Qi Y, Ramko EB, Davidson MW, Jin Y, Ellisman MH, Tsien RY (2011) A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol 9:e1001041

    PubMed Central  PubMed  CAS  Google Scholar 

  • Sipieter F, Vandame P, Spriet C, Leray A, Vincent P, Trinel D, Bodart JF, Riquet FB, Héliot L (2013) From FRET imaging to practical methodology for kinase activity sensing in living cells. Prog Mol Biol Transl Sci 113:145–216. doi:10.1016/B978-0-12-386932-6.00005-3. PMID:23244791

  • Sipieter F, Ladik M, Vandenabeele P, Riquet F (2014) Shining light on cell death processes - a novel biosensor for necroptosis, a newly described cell death program. Biotechnol J. PMID:24390900

    Google Scholar 

  • Smith CC, Davidson SM, Lim SY, Simpkin JC, Hothersall JS, Yellon DM (2007) Necrostatin: a potentially novel cardioprotective agent? Cardiovasc Drugs Ther 21:227–233

    PubMed  CAS  Google Scholar 

  • Smith CV (1987) Evidence for participation of lipid peroxidation and iron in diquat-induced hepatic necrosis in vivo. Mol Pharmacol 32:417–422

    PubMed  CAS  Google Scholar 

  • Smith G, Glaser M, Perumal M, Nguyen QD, Shan B, Arstad E, Aboagye EO (2008) Design, synthesis, and biological characterization of a caspase 3/7 selective isatin labeled with 2-[18F]fluoroethylazide. J Med Chem 51:8057–8067

    PubMed  CAS  Google Scholar 

  • Sosna J, Voigt S, Mathieu S, Lange A, Thon L, Davarnia P, Herdegen T, Linkermann A, Rittger A, Chan FK, Kabelitz D, Schütze S, Adam D (2014) TNF-induced necroptosis and PARP-1-mediated necrosis represent distinct routes to programmed necrotic cell death. Cell Mol Life Sci 71:331–348

    Google Scholar 

  • Stennicke H, Renatus M, Meldal M, Salvesen G (2000) Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8. Biochem J 350(Pt 2):563–568

    PubMed Central  PubMed  CAS  Google Scholar 

  • Suffys P, Beyaert R, De Valck D, Vanhaesebroeck B, Van Roy F, Fiers W (1991) Tumour-necrosis-factor-mediated cytotoxicity is correlated with phospholipase-A2 activity, but not with arachidonic acid release per se. Eur J Biochem/FEBS 195:465–475

    CAS  Google Scholar 

  • Sun L, Wang H, Wang Z, He S, Chen S, Liao D, Wang L, Yan J, Liu W, Lei X, Wang X (2012) Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 148:213–227

    PubMed  CAS  Google Scholar 

  • Takahashi N, Duprez L, Grootjans S, Cauwels A, Nerinckx W, DuHadaway JB, Goossens V, Roelandt R, Van Hauwermeiren F, Libert C, Declercq W, Callewaert N, Prendergast GC, Degterev A, Yuan J, Vandenabeele P (2012) Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models. Cell Death Dis 3:e437

    PubMed Central  PubMed  CAS  Google Scholar 

  • Thal SE, Zhu C, Thal SC, Blomgren K, Plesnila N (2011) Role of apoptosis inducing factor (AIF) for hippocampal neuronal cell death following global cerebral ischemia in mice. Neurosci Lett 499:1–3

    PubMed  CAS  Google Scholar 

  • Vanden Berghe T, Grootjans S, Goossens V, Dondelinger Y, Krysko DV, Takahashi N, Vandenabeele P (2013) Determination of apoptotic and necrotic cell death in vitro and in vivo. Methods 61:117–129

    PubMed  CAS  Google Scholar 

  • Vanden Berghe T, Kalai M, van Loo G, Declercq W, Vandenabeele P (2003) Disruption of HSP90 function reverts tumor necrosis factor-induced necrosis to apoptosis. J Biol Chem 278:5622–5629

    PubMed  CAS  Google Scholar 

  • Vanden Berghe T, Vanlangenakker N, Parthoens E, Deckers W, Devos M, Festjens N, Guerin CJ, Brunk UT, Declercq W, Vandenabeele P (2010) Necroptosis, necrosis and secondary necrosis converge on similar cellular disintegration features. Cell Death Differ 17:922–930

    PubMed  CAS  Google Scholar 

  • Vandenabeele P, Galluzzi L, Vanden Berghe T, Kroemer G (2010) Molecular mechanisms of necroptosis: an ordered cellular explosion. Nat Rev Mol Cell Biol 11:700–714

    PubMed  CAS  Google Scholar 

  • Vandenabeele P, Grootjans S, Callewaert N, Takahashi N (2013) Necrostatin-1 blocks both RIPK1 and IDO: consequences for the study of cell death in experimental disease models. Cell Death Differ 20:185–187

    PubMed Central  PubMed  CAS  Google Scholar 

  • Vanlangenakker N, Bertrand MJ, Bogaert P, Vandenabeele P, Vanden Berghe T (2011a) TNF-induced necroptosis in L929 cells is tightly regulated by multiple TNFR1 complex I and II members. Cell Death Dis 2:e230

    PubMed Central  PubMed  CAS  Google Scholar 

  • Vanlangenakker N, Vanden Berghe T, Bogaert P, Laukens B, Zobel K, Deshayes K, Vucic D, Fulda S, Vandenabeele P, Bertrand MJ (2011b) cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent reactive oxygen species production. Cell Death Differ 18:656–665

    PubMed Central  PubMed  CAS  Google Scholar 

  • Vanlangenakker N, Vanden Berghe T, Vandenabeele P (2012) Many stimuli pull the necrotic trigger, an overview. Cell Death Differ 19:75–86

    PubMed Central  PubMed  CAS  Google Scholar 

  • Vercammen D, Beyaert R, Denecker G, Goossens V, Van Loo G, Declercq W, Grooten J, Fiers W, Vandenabeele P (1998a) Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor. J Exp Med 187:1477–1485

    PubMed Central  PubMed  CAS  Google Scholar 

  • Vercammen D, Brouckaert G, Denecker G, Van de Craen M, Declercq W, Fiers W, Vandenabeele P (1998b) Dual signaling of the Fas receptor: initiation of both apoptotic and necrotic cell death pathways. J Exp Med 188:919–930

    PubMed Central  PubMed  CAS  Google Scholar 

  • Vince JE, Wong WW, Gentle I, Lawlor KE, Allam R, O’Reilly L, Mason K, Gross O, Ma S, Guarda G, Anderton H, Castillo R, Häcker G, Silke J, Tschopp J (2012) Inhibitor of Apoptosis Proteins Limit RIP3 Kinase-Dependent Interleukin-1 Activation. Immunity 36:215–227

    Google Scholar 

  • Wainwright M (2010) Dyes, trypanosomiasis and DNA: a historical and critical review. Biotech Histochem 85:341–354

    PubMed  CAS  Google Scholar 

  • Walker PR, Leblanc J, Smith B, Pandey S, Sikorska M (1999) Detection of DNA fragmentation and endonucleases in apoptosis. Methods 17:329–338

    PubMed  CAS  Google Scholar 

  • Wang K, Li J, Degterev A, Hsu E, Yuan J, Yuan C (2007) Structure-activity relationship analysis of a novel necroptosis inhibitor, Necrostatin-5. Bioorg Med Chem Lett 17:1455–1465

    PubMed  CAS  Google Scholar 

  • Wang L, Du F, Wang X (2008) TNF-alpha induces two distinct caspase-8 activation pathways. Cell 133:693–703

    PubMed  CAS  Google Scholar 

  • Wendland MF, Faustino J, West T, Manabat C, Holtzman DM, Vexler ZS (2008) Early diffusion-weighted MRI as a predictor of caspase-3 activation after hypoxic-ischemic insult in neonatal rodents. Stroke 39:1862–1868

    PubMed  Google Scholar 

  • Williams TM, Nyati S, Ross BD, Rehemtulla A (2013) Molecular Imaging of the ATM Kinase Activity. Int J Radiat Oncol Biol Phys 86:969–977

    PubMed  CAS  Google Scholar 

  • Wu P, Nicholls SB, Hardy JA (2013) A tunable, modular approach to fluorescent protease-activated reporters. Biophys J 104:1605–1614

    PubMed  PubMed Central  CAS  Google Scholar 

  • Wu Y, Wang D, Wang X, Wang Y, Ren F, Chang D, Chang Z, Jia B (2011) Caspase 3 is activated through caspase 8 instead of caspase 9 during H2O2-induced apoptosis in HeLa cells. Cell Physiol Biochem 27:539–546

    PubMed  CAS  Google Scholar 

  • Wyllie A (1981) Cell death: a new classification separating apoptosis from necrosis. In: Lockshin RA (ed) Cell death in biology and pathology. Chapman & Hall, London, pp 9–34

    Google Scholar 

  • Xu X, Chua CC, Zhang M, Geng D, Liu CF, Hamdy RC, Chua BH (2010) The role of PARP activation in glutamate-induced necroptosis in HT-22 cells. Brain Res 1343:206–212

    PubMed  CAS  Google Scholar 

  • Yamashima T (2000) Implication of cysteine proteases calpain, cathepsin and caspase in ischemic neuronal death of primates. Prog Neurobiol 62:273–295

    PubMed  CAS  Google Scholar 

  • Yazdanpanah B, Wiegmann K, Tchikov V, Krut O, Pongratz C, Schramm M, Kleinridders A, Wunderlich T, Kashkar H, Utermohlen O, Bruning JC, Schutze S, Kronke M (2009) Riboflavin kinase couples TNF receptor 1 to NADPH oxidase. Nature 460:1159–1163

    PubMed  CAS  Google Scholar 

  • Yoshioka A, Yamaya Y, Saiki S, Kanemoto M, Hirose G, Beesley J, Pleasure D (2000) Non-N-methyl-D-aspartate glutamate receptors mediate oxygen–glucose deprivation-induced oligodendroglial injury. Brain Res 854:207–215

    PubMed  CAS  Google Scholar 

  • You Z, Savitz SI, Yang J, Degterev A, Yuan J, Cuny GD, Moskowitz MA, Whalen MJ (2008) Necrostatin-1 reduces histopathology and improves functional outcome after controlled cortical impact in mice. J Cereb Blood Flow Metab 28:1564–1573

    PubMed Central  PubMed  CAS  Google Scholar 

  • Zhang DW, Shao J, Lin J, Zhang N, Lu BJ, Lin SC, Dong MQ, Han J (2009) RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science 325:332–336

    PubMed  CAS  Google Scholar 

  • Zhang J, Ye J, Altafaj A, Cardona M, Bahi N, Llovera M, Canas X, Cook SA, Comella JX, Sanchis D (2011) EndoG links Bnip3-induced mitochondrial damage and caspase-independent DNA fragmentation in ischemic cardiomyocytes. PLoS One 6:e17998

    PubMed Central  PubMed  CAS  Google Scholar 

  • Zhang Q, Raoof M, Chen Y, Sumi Y, Sursal T, Junger W, Brohi K, Itagaki K, Hauser CJ (2010) Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature 464:104–107

    PubMed Central  PubMed  CAS  Google Scholar 

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Grootjans, S., Goossens, V., Vandenabeele, P., Vanden Berghe, T. (2014). Methods to Study and Distinguish Necroptosis. In: Shen, HM., Vandenabeele, P. (eds) Necrotic Cell Death. Cell Death in Biology and Diseases. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4614-8220-8_18

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