Hi Jeannine, I adapted a manual platelet counting technique to lyse/elute the red cells when I was developing a whole blood method for Glanzmann's disease (also to avoid dual staining !) using a single marker (CD41). Try 3% Ammonium Oxalate, 20 vols to 1 vol of your whole blood prep and mix gently for circa 5 mins. It worked for me, but no guarantees that all markers will be unaffected - I didn't try anything other than CD's 41,42a and 61 and I worked only with citrated whole blood. Please let me know if this works for you. Wal Sharp NHS, UK -----Original Message----- From: Jeannine Navratil [mailto:jsn9@imap.pitt.edu] Sent: 29 March 2001 22:55 To: cyto-inbox Subject: Platelets Hello! I'm interested in surface staining platelets. I have a protocol (from Current Protocols in Cytometry) for staining whole blood, which uses thiazole orange coupled with a platelet-specific monoclonal antibody. I'd rather isolate the platelets from whole blood first, then stain them (so that I can avoid having to do multicolor analysis). It seems to be relatively simple to isolate platelets from plasma, but I've had trouble with them forming clumps. I used a wash buffer of PBS, 1% FBS, and 15mM EDTA. Possibly my concentration of EDTA was too low? Has anyone worked with platelets who can offer a few hints?? Thanks! Jeannine Navratil University of Pittsburgh Arthritis Institute
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