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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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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
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