Journal
METHODS
Volume 61, Issue 2, Pages 156-164Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2013.04.006
Keywords
Mitochondria; Apoptosis; BCL-2; Flow Cytometry; Cancer; Chemotherapy
Funding
- NIH [F31 CA150562, P01 CA139980, R01 CA129974]
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Rapid analysis of a cell's propensity to undergo apoptosis through the mitochondrial pathway is hindered by the complex network of interactions between more than fifteen known members of the BCL2 family that govern the decision to undergo mitochondrial apoptosis, and measurement of protein levels alone fails to account for critical interactions between the proteins. To address this issue, we have developed two functional assays for same-day analysis of cell lines or primary tissue samples. Using defined inputs in the form of peptides derived primarily from the BH3 domains of pro-apoptotic members of the BCL2 family, we invoke a response in the mitochondria in the form of mitochondrial outer membrane permeabilization measured indirectly using potential sensitive dyes. BH3 profiling can be applied to any viable single cell suspension and provides a response from the sum total of all known and unknown interactions within the BCL2 family for each stimulus, and the pattern of response can provide both a cell's propensity towards mitochondrial apoptosis, or 'priming', as well as indicate dependencies on specific anti-apoptotic proteins. Described here are optimized conditions for both plate-based and FACS-based BH3 profiling for homogeneous and heterogeneous samples. (C) 2013 Elsevier Inc. All rights reserved.
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