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SEP 15, 2015 7:00 AM PDT

WEBINAR: Less False Negatives: Quantifying Cell Viability by Simultaneous Triple Staining

Speakers
  • Research Scientist, Kroemer Lab, Paris, France
    Biography
      Oliver Kepp received his Ph.D. in 2006 from the Humboldt University of Berlin and the Max Planck Institute for Infection Biology in Berlin, Germany. He is currently a research scientist in the laboratory of Guido Kroemer, where he investigates several aspects of immunogenic cell death, focusing on systems biology approaches.
    • Research Engineer, Institut Gustave Roussy, Villejuif, France, Kroemer Lab, Paris, France
      Biography
        Allan Sauvat received his engineer's degree in 2011 from the Institut Supérieur de BioSciences in Paris, France. He is now a research engineer on the BioCell platform in Villejuif, France, where is focusing on HTS assay and software development for systems biology.

      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.

      Learning Objectives:
      • Learn about the protocol developed 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)
      • Learn how 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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