I can only provide some experiences with P-selectin expression. The epitope recognized by many of the anti-P-selectin antibodies are stable to formalins but labile to alcohols. Paraformaldehyde fixation should work, but may result in a mixture of permeablized and non-permablized cells. Hence the evaluation could be a mixture of internal and external P-selectin expression. I assume you are interested in external expression only? You may be able to gate the external expression (it will be much lower level than internal expression). I suggest you may want to look at the cells under a fluorescent microscope before doing flow on them to get an idea of how many have surface or cytoplasmic staining. Another problem is that almost any in vitro handling of platelets can activate them and cause surface expression of P-selectin on a variable percentage of platelets. You might want to practice and validate the handling of of normal samples including re-evaluation at different timepoints. Flow cytometry evaluation of P-selectin expression on platelets can be done but it can be tricky. Carol W. Johnson, DVM PhD Sr. Research Veterinary Pathologist Pharmacia Kalamazoo, MI William Ross <wgr8w@virginia.edu> on 03/31/2000 09:56:57 AM To: cyto-inbox cc: (bcc: Carol W Johnson/USKZO/PNU) Subject: platelet fixation does anyone no of a good platelet fixation procedure for the study of selectin expression? It is a study of myocardial infarction patients and timing the heart attacks is difficult and we're trying to fix the purified prep for later analysis. thanks in advance. Bill Ross William (Bill) Ross Director - FACS Core Facility MR-4, Box G005 University of Virginia School of Medicine Charlottesville, VA 22908 office (804) 982-1586 fax (804) 924-1221 e-mail:wgr8w@virginia.edu
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