Hello Michael, You're right, the three color method is sensitive to fixative and buffers. The CD61 and 62 part are reasonably well-behaved with only normal care about antibodies binding non-specifically based on optimal concentration and roughly physiological pH/salt conditions. The epitope recognized by Pac-1 is more delicate. You should have received BD's 3-Color Analysis of Platelet Activation procedure, which describes a fair bit of work optimizing the assay, and has some recommendations about specificity controls particular to Pac-1. We do not have experience using heparinized blood since we worried about heparin's tendency to activate platelets. Nearly all our work is done with citrated blood, which chelates Ca++ enough to block aggregation, but leaves enough around (I'm told about 50uM, but haven't measured it) so that we can still get activation with common agonists like ADP, TRAP, PMA or epinephrine. Pre-fixation has little effect on CD61 or CD62P signals, however PAC-1 binding is greatly reduced if platelets are fixed before staining (Shattil et al., 1987 Blood 70:307-315). You can certainly post-fix with 1% p-form in PBS. Not surprisingly, PAC-1 binding is sensitive to buffers that affect Ca+ levels (SJ Shattil et al. 1985, J Biol Chem 260,11107), pH and the amount of azide present during the staining reaction of the live platelets (our observations). And if you're new to platelet biology, be careful to isolate them without artificially activating them. Again, the BD procedure tells more about it. Glad to hear from anyone using the stuff.... Happy clotting, unclejack
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