High-throughput live cell imaging of apoptosis.
Apoptosis is important for embryonic development, tissue homeostasis, and removal of cells with (potentially transforming) DNA lesions or other types of injuries. Functional genomics screens performed to unravel apoptotic signaling cascades in the context of toxicant-induced cell injury commonly use apoptosis as an end-point. Here, a method to detect the accumulation of apoptotic cells in real time that is well suited for high-throughput screens is described. The method uses automated microscopy in a 96-well format setting to visualize binding of fluorescent annexin V to the outer membrane leaflet of apoptotic cells. The automated image acquisition is followed by quantitative analysis using bioinformatics software. A protocol for each of the steps in this kinetic method is described, which includes the caspase-dependent apoptotic response to toxic compounds in multiple cell types and demonstrates that RNAi-based gene silencing of candidate apoptotic regulators affects the apoptosis kinetics as expected. This protocol will be useful for functional genomics as well as chemical (drug) screens.
Puigvert
J C
JC
Leiden University, Leiden, The Netherlands.
de Bont
Hans
H
van de Water
Bob
B
Danen
Erik H J
EH
D000818:Animals; D017304:Annexin A5 / Q000032:analysis / Q000378:metabolism; D017209:Apoptosis; D002477:Cells / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*
D017304:Annexin A5