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 (FITC-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.
Jayaraman
Sundararajan
S
Department of Pathology, University of Miami School of Medicine, Miami, Florida 33136, USA. sjayaram@med.miami.edu
D000590:Amino Acid Chloromethyl Ketones / Q000737:chemistry / Q000494:pharmacology*; D017209:Apoptosis; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002470:Cell Survival; D053938:DNA Fragmentation; D004273:DNA, Neoplasm / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D004587:Electrophoresis, Agar Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D006801:Humans; D019169:Jurkat Cells; D011419:Propidium / Q000378:metabolism
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D004273:DNA, Neoplasm; D004791:Enzyme Inhibitors; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D011419:Propidium; D020169:Caspases; D016650:Fluorescein-5-isothiocyanate