Detection of Apoptotic Versus Autophagic Cell Death by Flow Cytometry.
Different modes of regulated cell death (RCD) can be initiated by distinct molecular machineries and their morphological manifestations can be difficult to discriminate. Moreover, cells responding to stress often activate an adaptive response centered around autophagy, and whether such a response is cytoprotective or cytotoxic cannot be predicted based on morphological parameters only. Molecular definitions are therefore important to understand various RCD subroutines from a mechanistic perspective. In vitro, various forms of RCD including apoptosis and autophagic cell death can be easily discriminated from each other with assays that involve chemical or pharmacological interventions targeting key components of either pathway. Here, we detail a straightforward method to discriminate apoptosis from autophagic cell death by flow cytometry, based on the broad-spectrum caspase inhibitor Z-VAD-fmk and the genetic inhibition of ATG5.
Gustave Roussy Cancer Campus, Villejuif, France.|Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, 15, rue de l'Ecole de Médecine, 75006, Paris, France.|INSERM, U1138, Paris, France.|Faculté de Medicine, Université Paris Saclay/Paris XI, Le Kremlin-Bicêtre, France.|Université Paris Descartes/Paris V, Sorbonne Paris Cité, Paris, France.
Valentina
V
Sica
Gustave Roussy Cancer Campus, Villejuif, France.|Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, 15, rue de l'Ecole de Médecine, 75006, Paris, France.|INSERM, U1138, Paris, France.|Université Paris Descartes/Paris V, Sorbonne Paris Cité, Paris, France.|Université Pierre et Marie Curie/Paris VI, Paris, France.
M Chiara
MC
Maiuri
Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, 15, rue de l'Ecole de Médecine, 75006, Paris, France. kroemer@orange.fr.|INSERM, U1138, Paris, France. kroemer@orange.fr.|Université Paris Descartes/Paris V, Sorbonne Paris Cité, Paris, France. kroemer@orange.fr.|Université Pierre et Marie Curie/Paris VI, Paris, France. kroemer@orange.fr.|Pôle de Biologie, Hôpital Européen Georges Pompidou AP-HP, Paris, France. kroemer@orange.fr.|Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France. kroemer@orange.fr.|Department of Women's and Children's Health, Karolinska University Hospital, Stockholm, Sweden. kroemer@orange.fr.
Guido
G
Kroemer
Gustave Roussy Cancer Campus, Villejuif, France. deadoc@vodafone.it.|Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, 15, rue de l'Ecole de Médecine, 75006, Paris, France. deadoc@vodafone.it.|INSERM, U1138, Paris, France. deadoc@vodafone.it.|Université Paris Descartes/Paris V, Sorbonne Paris Cité, Paris, France. deadoc@vodafone.it.|Université Pierre et Marie Curie/Paris VI, Paris, France. deadoc@vodafone.it.
Lorenzo
L
Galluzzi
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects; D000071187:Autophagy-Related Protein 5 / Q000037:antagonists & inhibitors; D061945:Caspase Inhibitors / Q000494:pharmacology; D045744:Cell Line, Tumor; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans
C502778:ATG5 protein, human; D000590:Amino Acid Chloromethyl Ketones; D000071187:Autophagy-Related Protein 5; D061945:Caspase Inhibitors; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone
Autophagy; Immunogenic cell death; Mitochondrial outer membrane permeabilization; Mitochondrial permeability transition; Necroptosis; Necrosis