Oncotarget

Research Papers:

Quantification of cellular viability by automated microscopy and flow cytometry

Allan Sauvat, Yidan Wang, Florian Segura, Sabrina Spaggiari, Kevin Müller, Heng Zhou, Lorenzo Galluzzi, Oliver Kepp, _ Guido Kroemer

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Oncotarget. 2015; 6:9467-9475. https://doi.org/10.18632/oncotarget.3266

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Abstract

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

Correspondence to:

Oliver Kepp, e-mail: oliver.kepp@gustaveroussy.fr

Guido Kroemer, e-mail: kroemer@orange.fr

Keywords: apoptosis, necrosis, high-throughput screening, drug discovery

Received: January 09, 2015     Accepted: January 31, 2015     Published: March 25, 2015

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.


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