Quantification of cellular viability by automated microscopy and flow cytometry

摘要

// Allan Sauvat 1, 2, 3 , Yidan Wang 1, 2, 3, 4 , Florian Segura 1, 2, 3 , Sabrina Spaggiari 1, 2, 3 , Kevin Müller 1, 2, 3 , Heng Zhou 1, 2, 3, 4 , Lorenzo Galluzzi 1, 2, 5, 6 , Oliver Kepp 1, 2, 3 , Guido Kroemer 1, 2, 3, 6, 7 1 Equipe 11 labellisée par la Ligue Nationale Contre le Cancer, Centre de Recherche des Cordeliers, Paris, France 2 INSERM, U1138, Paris, France 3 Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France 4 Faculté de Medecine, Université Paris-Sud, Le Kremlin-Bicêtre, France 5 Gustave Roussy Cancer Campus, Villejuif, France 6 Faculté de Medecine, Université Paris Descartes, Sorbonne Paris Cité, Paris, France 7 Pôle de Biologie, Hopitâl Européen George Pompidou, AP-HP, Paris, France Correspondence to: 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 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 DiOC 6 (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.