John the best way I know to recover neutrophils is to lay 0.5 ml peripheral blood onto a 1 ml ficoll 1.077 and let motionless for 15 minutes : you will have a clear pellet containing all WBC and the platelets (by the way this is a quick and simple way to obtain very rich plt specimens); this tecnique do not activate the PMN at all. Regarding the permeabilization I think that Triton X-100 (working concentration = 0.1 %) is the best item you can use Finally I think that Fab FITC IgG , as used by me to detect anti-platelets autoantibosies , is the right way to detect the target you need. Best regards Edoardo Colombo MD Director Flow Lab Dept. of Pathology Valduce Hospital COMO , Italy coledoco@tin.it
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