iBH3: simple, fixable BH3 profiling to determine apoptotic priming in primary tissue by flow cytometry.

Dysregulation of the mitochondrial pathway of apoptosis, controlled by the BCL-2 family of proteins, leads to disease states including cancer. Rapid analysis of a cell's dependency on the BCL-2 family of proteins is hindered by the complex interactions of more than a dozen proteins. Transcript or even protein levels are therefore generally insufficient to predict a cell's response to perturbations like chemotherapy. Previously, we developed the JC-1 BH3 method to provide a same day functional assay to assess a cell's propensity to undergo apoptosis and demonstrated its utility in predicting response to chemotherapy. We have now improved upon these methods to create a robust assay amenable to high throughput platforms using cytochrome c retention in formaldehyde fixed cells to remove the time sensitivity of JC-1 potential measurements. BH3 profiling by intracellular staining (iBH3) is suitable for 96- and 384-well formats, and can be used to directly screen candidate BH3-mimetic compounds for activity. When used as the final component of dynamic BH3 profiling (DBP), which uses a drug pretreatment prior to iBH3 to assess the change in profile due to treatment, it can predict the response of cells to chemotherapy days before they show signs of death.