To the ongoing discussion on the use of fluorochrome-labeled protease inhibitors to detect activated caspases let me add a note about our experience. First of all, the use of labeled inhibitors that bind to active centers of proteases has long history. In the 60's this approach was applied to detect active proteases e.g. in mast cells with radioisotope-tagged protease inhibitors which were then traced by autoradiography (see Nature, 213, 1198-1203, 1967). The essence of the methodology is based on covalent binding of the inhibitor (usually at 1:1 stoichiometry) to the active center of the enzyme. In the case of caspases one of several ligands that may specifically bind to their active center is a fluorochrome labeled fluoromethyl ketone-peptide conjugate. Such ligands [e.g. benzyloxycaronylvalylalanylaspartic acid fluoromethyl ketone (FAM-VAD-FMK), or FITC-VAD-FMK] have recently become comercially available. The first is available from Promega, Madison, WI, the second from Intergen Co., Purchase, N.Y. These ketone reagents penetrate through the plasma membrane of live cells and are relatively nontoxic to the cell. Their irreversible binding to active centers of the caspases ensures that only the cells with the activated enzymase become labeled. Because these inhibitors consist of three amino acid recognition sequence instead of four (four amino acid recognition is typical for individual caspases) they are rather generic for most caspases than specific for the particular one. We have experience with the reagent offered by Intergen. This reagent performs very well in two model systems of apoptosis, one of HL-60 cells treated with camptothecin (apoptosis induced via mitochondrial pathway) and another, with the same cells treated with TNF alpha plus cycloheximide (death ligand iduced apoptosis). We did measure cell fluorescence by laser scanning cytometry and confirmed, by cell re-location and morphologic examination, that that the cells labeled with FAM-VAD-FMK were apoptotic (manuscript in preparation). We also have a good experience with the antibody that detects the product of PARP cleavage that is offered by Promega (Exp. Cell Res., 255, 125-132, 2000). Zbigniew Darzynkiewicz ]
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