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High‐throughput in vivo quantification of apoptotic induction in yeast liquid culture (762.5)
Author(s) -
Luo Yi,
Schwotzer Erica,
Chirigos Allison,
Whitworth Gregg
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.762.5
Subject(s) - apoptosis , fragmentation (computing) , mutant , fluorescence microscope , yeast , fluorescence , microbiology and biotechnology , dna fragmentation , fluorescence in situ hybridization , biology , ex vivo , chemistry , biophysics , programmed cell death , biochemistry , in vitro , gene , chromosome , ecology , physics , quantum mechanics
We have developed a novel technique that allows for high‐throughput quantification of apoptotic induction in yeast cells growing in batch liquid culture. Our protocol combines cell permeable fluorescent dyes which probe molecular events associated with apoptosis (ROS species release from the mitochondria; nuclear DNA fragmentation) with 96‐well format to allow for the continuous measurement of both growth rate and apoptotic induction. As expected, background fluorescence intensity is much higher than in optimized microscopy‐based procedures. However, we see a significant shift in fluorescence intensity in cultures in which apoptosis has been induced by H2O2 or cation toxicity, compared to control un‐induced cultures. Importantly, this intensity shift is lost in mutants that fail to induce apoptosis under these conditions (ex. aif1Δ). We will describe the quantitative relationship between measurements made in batch culture and apoptotic rate measured by visualizing apoptosis‐specific stains using scanning confocal microscopy. We are currently leveraging the high‐throughput nature of this new assay to assemble a comprehensive dataset describing the quantitative response of yeast to dozens of apoptotic stress conditions across hundreds of mutant strains. Grant Funding Source : EJS, AJC, YL & GW supported by the HHMI Fellows Program & Grant Initiative; GW by Lenfest Grants.