The analysis of cytochrome c release is important in determining the commitment to apoptosis in model signaling systems. Cytochrome c is encoded by a nuclear gene and translated as apocytochrome c. Upon induction of apoptosis, holocytochrome c is released from mitochondria and associates with Apaf1 and procaspase-9, forming the "apoptosome". Apoptosome formation results in activation of caspase-9, which is responsible for activation of the proteolytic caspase cascade and the ultimate demise of the cell. The Calbiochem® InnoCyte™ Flow Cytometric Cytochrome c Release Kit (Cat. No. CBA077) relies on the selective permeabilization of the cellular membrane for release of cytosolic components while leaving the mitochondrial membrane intact. Viable cells will display mitochondrial staining of cytochrome c while cells committed to the apoptotic process do not stain since they release cytochrome c from the mitochondria to the cytosol. Cytochrome c is detected with a specific monoclonal antibody to cytochrome c and a FITC-labeled secondary antibody either by flow cytometry or fluorescent microscopy. |
