Method for monitoring of mitochondrial cytochrome c release during cell death: Immunodetection of cytochrome c by flow cytometry after selective permeabilization of the plasma membrane

Background: Cytochrome c release from mitochondria to cytosol is a hallmark of apoptosis and is used to characterize the mitochondria‐dependent pathway of this type of cell death. Techniques currently used to measure cytochrome c release, Western blot and fluorescence microscopy of immunolabeled cells, are time‐consuming and inaccurate, and the latter is still limited by sample size. Methods: We developed a rapid and reliable technique to detect cytochrome c release during drug‐induced apoptosis, using flow cytometry. Plasma membrane of apoptotic HL‐60 cells and thymocytes, treated with staurosporine and dexamethasone, respectively, were selectively permeabilized by digitonin at a low concentration. The released cytochrome c was quickly washed out from cells and that which remained in the mitochondria was immunolabeled after fixing the cells. Results: The fraction of cells that retained their mitochondrial cytochrome c, or the highly fluorescent cells, gradually decreased so that after 4–8 h of drug treatment almost all the cells lost their cytochrome c and emerged as a population of low fluorescent cells. This was confirmed by parallel fluorescence microscopy of cells immunolabeled for cytochrome c. Conclusions: This technique allows the analysis of cytochrome c release from mitochondria of a large number of apoptotic cells in a short period of time and is proposed as an alternative to the methods currently used for this same purpose. © 2006 International Society for Analytical Cytology