Quantification of cellular viability by automated microscopy and flow cytometry
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Allan Sauvat1,2,3, Yidan Wang1,2,3,4, Florian Segura1,2,3, Sabrina Spaggiari1,2,3, Kevin Müller1,2,3, Heng Zhou1,2,3,4, Lorenzo Galluzzi1,2,5,6, Oliver Kepp1,2,3, Guido Kroemer1,2,3,6,7
1Equipe 11 labellisée par la Ligue Nationale Contre le Cancer, Centre de Recherche des Cordeliers, Paris, France
2INSERM, U1138, Paris, France
3Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France
4Faculté de Medecine, Université Paris-Sud, Le Kremlin-Bicêtre, France
5Gustave Roussy Cancer Campus, Villejuif, France
6Faculté de Medecine, Université Paris Descartes, Sorbonne Paris Cité, Paris, France
7Pôle de Biologie, Hopitâl Européen George Pompidou, AP-HP, Paris, France
Oliver Kepp, e-mail: [email protected]
Guido Kroemer, e-mail: [email protected]
Keywords: apoptosis, necrosis, high-throughput screening, drug discovery
Received: January 09, 2015 Accepted: January 31, 2015 Published: March 25, 2015
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.
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