Figure 4.
Dynamic iBH3 profiling. A single-cell suspension is obtained from a cell line or primary cell sample. These cells are exposed to apoptosis-inducing drugs of interest for a short period of time. Cells are then gently permeabilized using digitonin to allow BH3 peptide uptake. Apoptotic priming is measured via cytochrome C retention using flow cytometry. By comparing the retained cytochrome C in nontreated vs treated cells, drug efficacy can be determined. The higher the release of cytochrome C the more effective the drug. This assay can be further used to measure potential shifts in prosurvival protein dependency.