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Cell Death Dis. 2016 Dec 1;7(12):e2493. doi: 10.1038/cddis.2016.332.

Robust high-throughput kinetic analysis of apoptosis with real-time high-content live-cell imaging.

Gelles JD1,2,3, Chipuk JE1,2,3,4,5.

Author information

1
Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, USA.
2
The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, USA.
3
The Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, USA.
4
Department of Dermatology, Icahn School of Medicine at Mount Sinai, New York, USA.
5
The Diabetes, Obesity, and Metabolism Institute, Icahn School of Medicine at Mount Sinai, New York, USA.

Abstract

Quantitative and kinetic analyses of apoptotic cell death are integral components of exploring cell biology, measuring cellular stress responses, and performing high-throughput genomic/RNAi/drug screens. Here, we present a detailed method that integrates robust kinetic real-time high-content imaging with Annexin V labelling to provide a highly sensitive, accurate, simple and zero-handling approach to quantify extrinsic and intrinsic inducers of apoptosis. The sensitivity of this non-toxic method outperforms previous high-throughput methodologies using viability dyes or caspase-activation reporters. This method also incorporates a multiplex adaptation to integrate variability in cell number due to treatment-induced proliferation changes and the detachment of dying cells. Compared to Annexin V detection by flow cytometry, this method is 10-fold more sensitive, eliminates extensive sample handling and processing, and provides real-time kinetics of apoptosis at both single-cell and population-level resolutions.

PMID:
27906190
PMCID:
PMC5261025
DOI:
10.1038/cddis.2016.332
[Indexed for MEDLINE]
Free PMC Article

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