From: William Magner (WMAGNER@atlas.niaid.nih.gov)
Date: Thu Jun 18 1998 - 17:34:18 EST
Hi Andy and other list members, I have used the Pharmingen kit and can report that, after some effort, I am happy with the results. I would be happy to help others avoid some of the problems we had. I've put together some notes that I can email to anyone who would like them or, if there is enough interest, I can just post them to the list. Briefly, we were successful with the caspase assay (in combination with surface staining) on a FACS Vantage with excitation at 380 nm and an emission filter at 455/20 nm. UV laser power was 60 mW (our PMT was 534). All data were saved for analysis. In our hands, the most critical parameters were the DEVD-AMC incubation time and temperature - minimum of 1 hour at 37C. Controls are extremely important since the AMC shift is relatively small. We used a Streptavidin-AMC for surface labeling controls to see maximal shifts. The caspase inhibited controls are also critical for comparison and worked well in our hands. The gating for data analysis is also important in that many of the caspase positive cells are lost with our standard (tight) FSC/SSC and PI gates. For surface staining in conjunction with caspase activity, the surface labeling must be done completely before addition of the caspase substrate therefore azide must be avoided in the surface staining antibodies. We were concerned about loss of surface staining during the 37C incubation but, for our markers, it was not a problem. Best wishes, Bill Magner William J. Magner, Ph.D. Senior Staff Fellow NIH/NIAID/OSD/SBS wmagner@nih.gov 301-496-3886 phone 301-402-0284 fax
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