Abstract
Apoptosis (programmed cell death) is activated by a wide variety of cellular stresses or insults and is vital for proper mammalian development as well as the maintenance of organismal homeostasis. The apoptosis pathway is also engaged by many common types of anticancer therapies and ionizing radiation, which contributes to the regressions of tumors or the toxic side effects of treatment. Due to the importance of maintaining healthy cell survival or the efficient clearance of cancer cells, the BH3 profiling assay was developed to functionally measure the state of the apoptosis pathway in any given cells. This assay involves the exposure of cellular mitochondria, where the BCL-2 family of proteins resides and controls the commitment to apoptosis, to proapoptotic BH3 peptides that mimic the activity of endogenous proapoptotic proteins. By using either activator or sensitizer peptides, the level of mitochondrial apoptotic priming (proximity to the threshold at which a cell commits to cell death) or dependence on prosurvival BCL-2 family proteins can be determined. Described here are two methods of BH3 profiling that can enable a user to make these functional measurements, which can be useful for predicting cellular responses to proapoptotic stressors or therapeutics (BH3 mimetics) that inhibit the activity of prosurvival proteins.