Interactions of fluorochrome‐labeled caspase inhibitors with apoptotic cells: A caution in data interpretation

Abstract

FLICA are specific and convenient to use markers of apoptotic cells and they detect very early events of apoptosis associated with caspases activation. Assays that combine their binding with either the loss of mitochondrial potential or with exclusion of PI as a probe of plasma membrane integrity, distinguish sequential stages of apoptosis and are particularly useful to differentiate between apoptosis and necrosis. Our results conform with the published data that unlabeled caspase inhibitors, when added after induction of apoptosis, cannot prevent activation of caspases detected by binding of biotinylated inhibitors or by cleavage of fluorogenic substrates. While FLICA binding by apoptotic cells most likely is a consequence of caspase activation, these binding events may also involve other or additional mechanisms than simply their specific attachment to the active enzyme centers of caspases.