Dr. Salinas inquires about caspase-8 assay by flow cytometry. The most specific approach would be to use Ab that reacts with the activated form of caspase-8 but not with pro-caspase-8. Although large number of caspase-8 Ab are commercially available, I have not seen yet the published data showing the use of Ab to detect activation of caspase-8 by flow cytometry. Abs against activated caspase-3 and -9 are listed in some catalogues. Alternative approach is to use either caspase specific fluorogenic substrate or fluorochrome-labeled inhibitor (FLICA), with the LEDT peptide sequence. We observed, however, that specificity of most substrates and inhibitors is not as great as advertised. Perhaps this is a reflection of the fact that to label the enzyme in live cells the substrates or inhibitore are being used at two to three orders of magnitude higher concentration than their binding association (Km). Because of that we do not know what is their effective local concentration within the cells at the site of activated caspase. Neither we know whether Km (and peptide specificity) of a particular caspase in situ is the same as of the isolated enzyme (which served to test the specificity). One has to be careful, therefore, drawing conclusions about specificity of such reagents. Zbigniew Darzynkiewicz Brander Cancer Research Institute New York Medical College 19 Bradhurst Ave. Hawthorne, NY 10532 tel: 914-347-2801 fax: 914-347-2804 http://www.geocities.com/z_darzynkiewicz
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