 |
 |
 |
 |
 |
A few other hints (for the new to platelets): washing and lysing
protocols can sometimes activate the platelets, as you may know, so
we try to avoid this. Not washing away excess Ab can lead to higher
nonspecific fluorescence, so antibodies should be titered to optimum
concentration per platelet number in your lab prior to establishing a
routine protocol. Always add negative control Ab at the same ug
amount as positive Ab, and verify the same isotype and molar ratio of
dye to protein. BD makes a good PE platelet neg CTL (cat # 340013)
that has reduced non-bound PE in the suspension, but we have used
others that seem to work as well. Also, try to avoid using EDTA as
the anticoagulant. GPIIbIIIa expression is Ca dependent to some
degree, as well as some activation markers. Na3Citrate is best.
Miller and Bode have shown that triggering on linear side scatter
(visualizing on log forward scatter) gives the best platelet
distribution.
I'm sure there are other opinions in the field, but the above has
worked well for us. I hope you find it useful.
|
|
|
|
|
|
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;
Web