Fig. 7. Annexin V-binding by differentiating C2C12 (A) and H9C2 (B) myoblasts does not require loss of mitochondrial inner membrane potential or caspase activity. Apoptotic myoblast are positive for annexin V-fluo (green) and show a diffuse labeling for CMXRos (A1,B1, arrowhead, red) and immunoreactivity for the anti-active caspase 3 antibody (A3,B3,B4, arrowhead, red). By contrast, differentiating C2C12 and H9C2 cells bind annexin V-fluo while having a granular labeling for CMXRos (A2,B2, arrow) and show no active caspase 3 protein (A4,B4, arrow). In line with the latter results, culturing cells in DM containing the caspase inhibitor zVAD-(OMe)-fmk at a dose of 100 µM blocks neither myotube formation (A5,B5, arrow) nor annexin V-fluo binding (A6,B6, arrow). These two figures, in which out-of-focus information has been removed by image restoration using deconvolution software, further illustrate the intracellular granular labeling of myoblasts with annexin V-fluo after longer incubation periods. Bars, 25 µm.