title: Robust high-throughput kinetic analysis of apoptosis with real-time high-content live-cell imaging.
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.
authors.affiliation: Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, USA.|The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, USA.|The Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, USA.
authors.forename: Jesse D
authors.initials: JD
authors.lastname: Gelles
authors.affiliation: Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, USA.|The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, USA.|The Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, USA.|Department of Dermatology, Icahn School of Medicine at Mount Sinai, New York, USA.|The Diabetes, Obesity, and Metabolism Institute, Icahn School of Medicine at Mount Sinai, New York, USA.
authors.forename: Jerry Edward
authors.initials: JE
authors.lastname: Chipuk
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival; D005452:Fluoresceins / Q000378:metabolism; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D021621:Imaging, Three-Dimensional; D007700:Kinetics; D051379:Mice; D011485:Protein Binding; D013194:Staining and Labeling; D013388:Succinimides / Q000378:metabolism
chemical_list: C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D017304:Annexin A5; D005452:Fluoresceins; D013388:Succinimides