Review Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes L Galluzzi1,2,3, SA Aaronson4, J Abrams5, ES Alnemri6, DW Andrews7, EH Baehrecke8, NG Bazan9, MV Blagosklonny10, K Blomgren11,12, C Borner13, DE Bredesen14,15, C Brenner16,17, M Castedo1,2,3, JA Cidlowski18, A Ciechanover19, GM Cohen20, V De Laurenzi21, R De Maria22,23, M Deshmukh24, BD Dynlacht25, WS El-Deiry26, RA Flavell27,28, S Fulda29, C Garrido30,31, P Golstein32,33,34, M-L Gougeon35, DR Green36, H Gronemeyer37,38,39, G Hajnoczky40, �� JM Hardwick41, MO Hengartner42, H Ichijo43, M Jaattela44, �� �� �� O Kepp1,2,3, A Kimchi45, DJ Klionsky46, RA Knight47, S Kornbluth48, S Kumar49, B Levine28,50, SA Lipton51,52,53,54, E Lugli55, F Madeo56, W Malorni57, J-CW Marine58,59, SJ Martin60, JP Medema61,62, P Mehlen63,64,65, G Melino20,66, UM Moll67,68,69, E Morselli1,2,3, S Nagata70, DW Nicholson71, P Nicotera20, G Nunez72, �� M Oren73, J Penninger74, S Pervaiz75,76,77, ME Peter78, M Piacentini79,80, JHM Prehn81, H Puthalakath82, GA Rabinovich83, R Rizzuto84, CMP Rodrigues85, DC Rubinsztein86, T Rudel87, L Scorrano88,89, H-U Simon90, H Steller28,91, J Tschopp92, Y Tsujimoto93, P Vandenabeele59,94, I Vitale1,2,3, KH Vousden95, RJ Youle96, J Yuan97, B Zhivotovsky98 and G Kroemer*,1,2,3 Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells. Cell Death and Differentiation (2009) 16, 1093���1107 doi:10.1038/cdd.2009.44 published online 17 April 2009 1INSERM, U848, F-94805 Villejuif, France 2Institut Gustave Roussy, F-94805 Villejuif, France 3Universite �� Paris Sud-XI, F-94805 Villejuif, France 4Department of Oncological Sciences, Mount Sinai School of Medicine, New York, NY 10029, USA 5Department of Cell Biology, UT Southwestern Medical Center, Dallas, TX 75390, USA 6Department of Biochemistry and Molecular Biology, Center for Apoptosis Research, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107-5587, USA 7Department of Biochemistry and Biomedical Sciences, McMaster University, L8N 3Z5 Hamilton, Canada 8Department of Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605-2324, USA 9Neuroscience Center of Excellence, School of Medicine, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA 10Roswell Park Cancer Institute, Buffalo, NY 14263, USA 11Center for Brain Repair and Rehabilitation, Institute of Neuroscience and Physiology, University of Gothenburg, SE-405 30 Gothenburg, Sweden 12Department of Pediatric Oncology, The Queen Silvia Children���s Hospital, SE-416 85 Gothenburg, Sweden 13Institute of Molecular Medicine and Cell Research (ZBMZ), Albert-Ludwigs-Universitat �� Freiburg, 79104 Freiburg, Germany 14Buck Institute for Age Research, Novato, CA 94945, USA 15University of California ��� San Francisco, San Francisco, CA 94143, USA 16University of Versailles/St Quentin, 78035 Versailles, France 17CNRS, UMR8159, 78035 Versailles, France 18National Institutes of Environmental Health Sciences, NIH, Duhram, NC 27709, USA 19Vascular and Tumor Biology Research Center, The Rappaport Faculty of Medicine, Technion ��� Israel Institute of Technology, 31096 Haifa, Israel 20Medical Research Council, Toxicology Unit, Leicester University, Leicester LE1 9HN, UK 21Dipartimento di Scienze Biomediche, Universita ` ���G. d���Annunzio��� Chieti-Pescara, 66100 Chieti, Italy 22Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanita, ` 00161 Rome, Italy 23Mediterranean Institute of Oncology, 95030 Catania, Italy 24Neuroscience Center, Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, NC 27599-7250, USA 25Department of Pathology, New York University School of Medicine, New York, NY 10016, USA 26Hematology-Oncology Division, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA 27Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520, USA 28Howard Hughes Medical Institute, Chevy Chase, MD 20815-6789, USA 29University Children���s Hospital, 89075 Ulm, Germany 30INSERM, UMR866, 21049 Dijon, France 31Faculty of Medicine and Pharmacy, University of Burgundy, 21049 Dijon, France 32INSERM, U631, 13288 Marseille, France 33CNRS, UMR6102, 13288 Marseille, France 34Centre d���Immunologie de Marseille-Luminy, Aix Marseille Universite, �� 13288 Marseille, France 35Institut Pasteur, Antiviral Immunity, Biotherapy and Vaccine Unit, 75015 Paris, France 36Department of Immunology, St. Jude Children0s Research Hospital, Memphis, TN 38105, USA 37Department of Cancer Biology ��� Institut de Genetique �� �� et de Biologie Moleculaire �� et Cellulaire, 67404 Illkirch, France 38CNRS, UMR7104, 67404 Illkirch, France 39INSERM, U964, 67404 Illkirch, France 40Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA 41Department of Pharmacology and Molecular Sciences, Johns Hopkins University, Baltimore, MD 21205, USA 42Institute of Molecular Biology, University of Zurich, 8057 Zurich, Switzerland 43Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan 44Danish Cancer Society, Department of Apoptosis, Institute of Cancer Biology, DK-2100 Copenhagen, Denmark 45Department of Molecular Genetics, Weizmann Institute of Science, 76100 Rehovot, Israel 46Life Sciences Institute and Department of Molecular, Cellular, and Developmental Biology and Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA 47Institute of Child Health, University College London, London WC1N 1EH, UK 48Duke University School of Medicine, Durham, NC 27710, USA 49Centre for Cancer Biology, Hanson Institute, Adelaide, South Australia 5000, Cell Death and Differentiation (2009) 16, 1093���1107 & 2009 Macmillan Publishers Limited All rights reserved 1350-9047/09 $32.00 www.nature.com/cdd

In multicellular organisms, the timely execution of programmed cell death is critical for numerous physiological processes including embryogenesis, post-embryonic development and adult tissue homeostasis. It is, therefore, not surprising that deregulated cell death is a common feature of a wide array of human diseases. On one hand, the unwarranted death of postmitotic cells constitutes one of the most important etiological determinants of acute and chronic pathologies including (but not limited to) ischemic, toxic, neurodegenera- tive and infectious syndromes. Conversely, disabled cell death is frequently associated with hyperproliferative conditions such as autoimmune diseases and cancer. Several well-established and experimental therapies target the molecular mechanisms of cell death, either to prevent the demise of cells that cannot be replaced, or to facilitate the elimination of supernumerary and/or ectopic cells.1 Thus, the precise characterization of the molecular machinery of cell death constitutes a major challenge for present and future research, which has already and will continue to have tremendous repercussions on the development of novel therapeutic approaches. The first and most important question that any researcher who studies cellular demise needs to answer is: when is a cell ���dead���? Recently, the Nomenclature Committee on Cell Death (NCCD) has formulated several recommendations on the use of cell death-related terminology.2 Dying cells are engaged in a cascade of molecular events that is reversible until a first irreversible process takes place, and the ���point-of-no-return��� that delimits the frontier between a cell���s life and death has been trespassed. So far, a single molecular event that accounts for the point-of-no-return in the signaling cascades leading to cell death remains to be identified. Thus, the NCCD has proposed that a cell should be regarded as ���dead��� when (1) the cell has lost the integrity of its plasma membrane and/ or (2) the cell, including its nucleus, has undergone complete disintegration, and/or (3) its corpse (or its fragments) has been engulfed by a neighboring cell in vivo. In this context, another important issue is represented by the indisputable existence of numerous cell death modalities.2 Cell death represents a highly heterogeneous process that can follow the activation of distinct (although sometimes partially overlapping) biochemical cascades and can manifest with different morphological features. For instance, cells can die as they display an apoptotic morphology (which among other features is characterized by chromatin condensation, Australia 50Southwestern Medical Center, University of Texas, Dallas, TX 75390, USA 51Burnham Institute for Medical Research, La Jolla, CA 92037, USA 52The Salk Institute for Biological Studies, La Jolla, CA 92037, USA 53The Scripps Research Institute, La Jolla, CA 92037, USA 54Univerisity of California-San Diego, La Jolla, CA 92093, USA 55Immunotechnology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA 56Institute of Molecular Biosciences, University of Graz, 8010 Graz, Austria 57Department of Therapeutic Research and Medicines Evaluation, Section of Cell Aging and Degeneration, Istituto Superiore di Sanita, ` 00161 Rome, Italy 58Laboratory for Molecular Cancer Biology, VIB, 9052 Ghent, Belgium 59Department for Molecular Biology, Ghent University, 9052 Ghent, Belgium 60Department of Genetics, Trinity College, Dublin 2, Ireland 61Center for Experimental and Molecular Medicine, Academic Medical Center, 1105 AZ Amsterdam, The Netherlands 62University of Amsterdam, 1012 ZA Amsterdam, The Netherlands 63Apoptosis, Cancer, and Development Laboratory, Centre Leon �� Berard, 69008 Lyon, France 64CNRS, UMR5238, 69008 Lyon, France 65Universite �� de Lyon, 69008 Lyon, France 66Department of Experimental Medicine and Biochemical Sciences, University of Rome ���Tor Vergata���, 00133 Rome, Italy 67Department of Pathology, Stony Brook University, Stony Brook, NY 11794-8691, USA 68Department of Molecular Oncology, Gottingen �� Center of Molecular Biosciences, 37077 Gottingen, �� Germany 69Faculty of Medicine, University of Gottingen, �� 37077 Gottingen, �� Germany 70Department of Medical Chemistry, Graduate School of Medicine, University of Kyoto, Kyoto 606-8501, Japan 71Merck Research Laboratories, Rahway, NJ 07065-0900, USA 72University of Michigan Medical School, Ann Arbor, MI 48109, USA 73Department of Molecular Cell Biology, Weizmann Institute of Science, 76100 Rehovot, Israel 74Institute of Molecular Biotechnology of the Austrian Academy of Science, 1030 Vienna, Austria 75Department of Physiology, Yong Loo Lin School of Medicine, Graduate School for Integrative Sciences and Engineering, National University of Singapore, 117597 Singapore 76Singapore-MIT Alliance, National University of Singapore, 117576 Singapore 77Duke-NUS Graduate Medical School, 169547 Singapore 78Ben May Department for Cancer Research, University of Chicago, Chicago, IL 60637, USA 79Laboratory of Cell Biology, National Institute for Infectious Diseases IRCCS ���L. Spallanzani���, 00149 Rome, Italy 80Department of Biology, University of Rome ���Tor Vergata���, 00133 Rome, Italy 81Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin 2, Ireland 82Department of Biochemistry, La Trobe University, 3086 Victoria, Australia 83Laboratorio de Inmunopatolog��a,�� Instituto de Biolog��a �� y Medicina Experimental (IBYME- CONICET), C1428 Buenos Aires, Argentina 84Department of Biomedical Sciences, University of Padova, 35121 Padova, Italy 85iMed.UL, Faculty of Pharmacy, University of Lisbon, 1649-003 Lisbon, Portugal 86Cambridge Institute for Medical Research, Cambridge CB2 0XY, UK 87Biocenter, University of Wurzburg, �� 97074 Wurzburg, �� Germany 88Department of Cell Physiology and Metabolism, University of Geneva Medical School, 1211 Geneva, Switzerland 89Dulbecco-Telethon Institute, Venetian Institute of Molecular Medicine, 35129 Padova, Italy 90Department of Pharmacology, University of Bern, 3010 Bern, Switzerland 91Laboratory of Apoptosis and Cancer Biology, The Rockefeller University, New York, NY 10065, USA 92Department of Biochemistry, University of Lausanne, 1066 Epalinges, Switzerland 93Department of Medical Genetics, Osaka University Medical School, Osaka 565-0871, Japan 94Department for Molecular Biomedical Research, VIB, 9052 Ghent, Belgium 95The Beatson Institute for Cancer Research, Glasgow G61 1BD, UK 96Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD 20892, USA 97Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA 98Institute of Environmental Medicine, Division of Toxicology, Karolinska Institute, SE- 171 77 Stockholm, Sweden *Corresponding author: G Kroemer, INSERM, U848, Institut Gustave Roussy, PR1, 39, rue Camille Desmoulins, F-94805 Villejuif, France. Tel: �� 33-1-4211-6046 Fax: �� 33-1-4211-6047 E-mail: kroemer@orange.fr Keywords: apoptosis caspases cytofluorometry immunofluorescence microscopy mitotic catastrophe necrosis Abbreviations: AIF, apoptosis-inducing factor AO, acridine orange CMXRos, chloromethyl-X-rosamine Cyt c, cytochrome c Dcm, mitochondrial transmembrane potential DAPI, 40,6-diamidino-2-phenylindole DiOC6(3), 3,30dihexiloxalocarbocyanine iodide EB, ethidium bromide ELISA, enzyme-linked immunosorbent assay GFP, green fluorescent protein H2DCFDA, 20,70-dichlorodihydrofluorescein diacetate HE, hydroethidine HPLC, high-pressure liquid chromatography HTS, high- throughput screening IMS, mitochondrial intermembrane space JC-1, 5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazolcarbocyanine iodide LDH, lactate dehydrogenase MOMP, mitochondrial outer membrane permeabilization MPT, mitochondrial permeability transition MS, mass spectrometry MTS, 3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide NCCD, Nomenclature Committee on Cell Death NMP, nuclear matrix protein NMR, proton nuclear magnetic resonance PI, propidium iodide TMRM, tetramethylrhodamine methyl ester TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling WST-1, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3- benzene disulfonate Received 12.3.09 accepted 17.3.09 Edited by G Melino published online 17.4.09 Monitoring cell death in higher eukaryotes L Galluzzi et al 1094 Cell Death and Differentiation

nuclear fragmentation and overall shrinkage of the cell) or a necrotic one (which is associated with a gain in cell volume, organellar swelling and disorganized dismantling of intracel- lular contents). Mixed cell death morphotypes characterized by both apoptotic and necrotic traits have also been described, which has led some investigators to suggest the existence of a ���continuum��� of cell death phenotypes, at least in specific experimental settings.3 Such morphological hetero- geneity frequently derives from the activation of separate executioner mechanisms. Thus, beyond merely encyclopedic intents, the correct classification of cell death into specific subroutines may be extremely important for its therapeutic implications. As an example, tumor cells are often resistant to chemotherapeutic regimens that induce apoptosis, but not to necrotic triggers. In this context, the induction of one specific cell death mode (i.e., necrosis), as opposed to another (i.e., apoptosis), would result in an obvious therapeutic advantage. The term ���autophagic cell death��� has been widely employed to indicate a type of cell death that is accompanied by massive vacuolization of the cytoplasm.2 However, the relationship between autophagy and cell death remains controversial.4,5 Multiple Drosophila melanogaster developmental scenarios (including involution of salivary glands, early oogenesis and removal of the extraembryonic tissue known as amnioserosa) provide in vivo evidence that cell death can be (at least partially) executed through autophagy.6���9 Consistent with these results, the knockout/knockdown of essential auto- phagy (atg) genes has been shown to protect cultured mammalian cells from some lethal inducers, at least in specific experimental settings.10 Still, more frequently, pharmacological and/or genetic inhibition of autophagy does not prevent cell death, and rather accelerates it.11,12 This suggests that although cell death can occur together with autophagy, the latter likely represents a prosurvival mechanism activated by dying cells in the attempt to cope with stress.11,12 As very detailed guidelines concerning the use and interpretation of assays for monitoring autophagy have been recently provided by Klionsky and colleagues,13 this topic will not be discussed further in the present review. Nowadays, dozens (if not hundreds) of methods are available for the detection of cell death-related parameters in vitro (in cell cultures), ex vivo (in explanted tissues and/or organs) and in vivo (in model organisms and/or humans Figure 1). Since the beginning of cell death research, this methodological collection has been evolving, driven by the technological innovation that has characterized the last decades. However, some of the classical methods to identify dead and dying cells (e.g., light microscopy-based techni- ques) continue to be largely employed by researchers (due to their simplicity and/or low cost), even though they may be rather nonspecific and, therefore, inappropriate in the majority of experimental settings. Conversely, the precise quantifica- tion of a single molecular process may be excessively specific, and also result in the over- and/or underestimation of cell death. Numerous methods to detect cell death can only be applied to a limited number of experimental settings, due to intrinsic features of the model system or technical limitations of the platform on which such protocols are implemented. Beyond obvious technical variations, the experimental procedures to identify dead and dying cells differ from one another with regard to (and hence may be classified according to) (1) specificity (i.e., some techniques selectively detect apoptosis-related phenomena, such as internucleosomal DNA cleavage, whereas others cannot discriminate between apoptotic and nonapoptotic cell death subroutines) (2) sensitivity (which is determined by the lower detection limit) (3) detection range (which relates to the upper detection limit) (4) precision (i.e., cell death-related parameters can be detected in a qualitative, semiquantitative or quantitative fashion) (5) throughput (which can be low, as for electron microscopy-based methods, standard, as for normal labora- tory practices, or high, as for automated procedures) (6) cell death stage (meaning that biochemical processes belong- ing either to the induction/initiation, integration/decision or execution/degradation phases of the cell death cascade can be specifically quantified) (7) cell death parameter (i.e., morphological versus biochemical) or (8) readout (which can be an end-point or a real-time measurement). Concerning specificity, a clear-cut distinction has to be made between ���general��� and ���cell death-type specific��� techniques. Although the former (e.g., vital dyes) can detect end-stage cell death irrespective of its type (most frequently by assessing the structural dismantling of dead cells and in particular plasma membrane breakdown), the latter (e.g., caspase activation assays) monitor processes that have been specifically, yet not exclusively, associated with a particular subroutine of cell death. This hierarchical subdivision reflects the correct experimental approach that should be used when studying cell death (see also ���Concluding remarks���). Irrespective of the possible categorization of the methods to detect cell death, standardized guidelines on their use and interpretation have never been formulated. Recently, Klionsky and colleagues have approached a similar issue concerning the techniques to detect autophagy.13 Along the lines of this work, we propose here a comparison of the most common methodologies to identify and quantify dead and dying cells, with particular emphasis on their relative advantages/draw- backs and on their suitability for specific versus common experimental scenarios. Light Microscopy, Electron Microscopy and (Immuno)cyto(histo)chemistry Visual inspection by light microscopy provides a rapid and inexpensive means to detect cell death in a generalized and rather nonspecific fashion. This can be done on living samples (in phase contrast mode, for instance, to monitor the conditions of cultured cells), or on fixation and staining of cytospins and/or histological sections. The most common cyto(histo)chemical protocols include Papanicolaou and Mayer���s hematoxylin/eosin (H&E) stains, both of which allow the visualization of multiple intracellular structures, and in particular of the nuclei. Thus, cells displaying morphological changes that normally are associated with cell death, such as pyknotic nuclei, membrane blebbing or swollen cytoplasm can be visualized. Still, these techniques are time consuming and operator dependent, and tend to underestimate the fraction of dead/dying cells. This is due to the fact that cells in the early phases of lethal cascades usually fail to display gross morphological modifications, and hence remain undetected Monitoring cell death in higher eukaryotes L Galluzzi et al 1095 Cell Death and Differentiation