Intracellular determination of activated caspases (IDAC) by flow cytometry using a pancaspase inhibitor labeled with FITC

Background: Flow cytometry-based methods of simultaneous detection of multiple activated caspases in single cells are of diagnostic value and remain to be fully explored. Methods: Genomic DNA fragmentation was determined by agarose gel electrophoresis and flow cytometry. Whole cells were incubated with the cell-permeable pancaspase inhibitor, FITC-Val-Ala-Asp-fluoro-methyl-Rerone (FITG VAD-FMK), and propidium iodide (PI) and analyzed for intracellular activated caspases and plasma membrane integrity, respectively, by flow cytometry. Results: Two distinct populations of apoptotic cells were identified by flow cytometry: an early apoptotic population indicated by FITC-VAD-FMK binding and the late apoptotic cells characterized by FITC-VAD-FMK staining and permeability to PI. The binding of FITC-VAD-FMK to apoptotic cells was time dependent and concurred with DNA fragmentation. Pretreatment with the pancaspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-methyl-fluoro-methyl-kerone (Z-VAD (OMe)-FMK), coordinately attenuated apoptosis induction and activation of caspases. The pancaspase inhibitor also competitively blocked the binding of FITC-VAD-FMK to early apoptotic cells in vitro. Significantly, the use of FITC-VAD-FMK permitted apoptosis determination in a number of cell lines including the caspase-3 gene-deleted MCF-7 responding to various apoptotic stimuli. Conclusions: Apoptosis detection based on FITC-VAD-FMK binding is specific and easily performed by flow cytometry in a variety of cells. This novel technique has implications for detecting apoptosis in pathophysiologic conditions. (C) 2003 Wiley-Liss, Inc.