title: Quantification of cellular viability by automated microscopy and flow cytometry.
abstract: Cellular viability is usually determined by measuring the capacity of cells to exclude vital dyes such as 4',6-diamidino-2-phenylindole (DAPI), or by assessing nuclear morphology with chromatinophilic plasma membrane-permeant dyes, such as Hoechst 33342. However, a fraction of cells that exclude DAPI or exhibit normal nuclear morphology have already lost mitochondrial functions and/or manifest massive activation of apoptotic caspases, and hence are irremediably committed to death. Here, we developed a protocol for the simultaneous detection of plasma membrane integrity (based on DAPI) or nuclear morphology (based on Hoechst 33342), mitochondrial functions (based on the mitochondrial transmembrane potential probe DiOC6(3)) and caspase activation (based on YO-PRO®-3, which can enter cells exclusively upon the caspase-mediated activation of pannexin 1 channels). This method, which allows for the precise quantification of dead, dying and healthy cells, can be implemented on epifluorescence microscopy or flow cytometry platforms and is compatible with a robotized, high-throughput workflow.
authors: Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France.|INSERM, U1138, Paris, France.|Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France.
authors: Allan
authors: A
authors: Sauvat
authors: Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France.|INSERM, U1138, Paris, France.|Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France.|Faculté de Medecine, Université Paris-Sud, Le Kremlin-Bicêtre, France.
authors: Yidan
authors: Y
authors: Wang
authors: Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France.|INSERM, U1138, Paris, France.|Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France.
authors: Florian
authors: F
authors: Segura
authors: Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France.|INSERM, U1138, Paris, France.|Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France.
authors: Sabrina
authors: S
authors: Spaggiari
authors: Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France.|INSERM, U1138, Paris, France.|Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France.
authors: Kevin
authors: K
authors: Müller
authors: Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France.|INSERM, U1138, Paris, France.|Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France.|Faculté de Medecine, Université Paris-Sud, Le Kremlin-Bicêtre, France.
authors: Heng
authors: H
authors: Zhou
authors: Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France.|INSERM, U1138, Paris, France.|Gustave Roussy Cancer Campus, Villejuif, France.|Faculté de Medecine, Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors: Lorenzo
authors: L
authors: Galluzzi
authors: Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France.|INSERM, U1138, Paris, France.|Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France.
authors: Oliver
authors: O
authors: Kepp
authors: Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France.|INSERM, U1138, Paris, France.|Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France.|Faculté de Medecine, Université Paris Descartes, Sorbonne Paris Cité, Paris, France.|Pôle de Biologie, Hopitâl Européen George Pompidou, AP-HP, Paris, France.
authors: Guido
authors: G
authors: Kroemer
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001562:Benzimidazoles / Q000032:analysis; D002232:Carbocyanines / Q000032:analysis / Q000378:metabolism; D002289:Carcinoma, Non-Small-Cell Lung / Q000473:pathology; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability; D002470:Cell Survival / Q000187:drug effects; D017630:Connexins / Q000378:metabolism; D004357:Drug Synergism; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007211:Indoles / Q000032:analysis / Q000378:metabolism; D008175:Lung Neoplasms / Q000473:pathology; D053078:Membrane Potential, Mitochondrial; D008856:Microscopy, Fluorescence / Q000379:methods*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009944:Organoplatinum Compounds / Q000494:pharmacology; D000077150:Oxaliplatin; D012371:Robotics; D013194:Staining and Labeling / Q000379:methods*; D019311:Staurosporine / Q000494:pharmacology; D057188:Workflow
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D001562:Benzimidazoles; D002232:Carbocyanines; D017630:Connexins; D005456:Fluorescent Dyes; D007211:Indoles; D009419:Nerve Tissue Proteins; D009944:Organoplatinum Compounds; C492764:PANX1 protein, human; C098830:TO-PRO-3; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D000077150:Oxaliplatin; C007293:DAPI; C007392:3,3'-dihexyl-2,2'-oxacarbocyanine; D020169:Caspases; D019311:Staurosporine; C017807:bisbenzimide ethoxide trihydrochloride
keywords: apoptosis; drug discovery; high-throughput screening; necrosis