I'm interested in developing a very simple protocol for measuring baseline P-selectin exposure on canine platelets. The assay needs to be run on research samples collected from veterinary patients and so the timing of sample collection is unpredictable and personnel may not always be available to immediately stain samples.
I've found a protocol on the BD website in which citrated whole blood is fixed immediately post-collection using cold 1% paraformaldehyde for 1-2 hours or more; washed, re-suspended and then stained for CD61 and CD62P.
Does anyone have experience of this protocol in any species? I'm particularly interested in 1) does the 1% fixation work to prevent spontaneous activation; (2) is the cold exposure a problem and 3) how well antibodies bind post fixation?
Thanks in advance
Unity
I can't answer specifically about the paraformaldehyde, but using citrated whole blood can completely stop activation at suffcient concentrations, chelating the calcium out of the blood. Without formaldehyde fixation, we have had to re-administer calcium to whole blood and PRP samples to allow for normal activation patterns. So, combining a light citrated with a fixating agent such as paraformaldehyde will probably do wonders for stopping any reactions, but I'll leave it up to others to tell you about whether the post-fixation binding can occur.
I have examined CD62P in both fixed then stained samples, as well as stained then fixed. I must say I would highly recommend the latter. Blood can be collected in citrated tubes as Juan mentioned, stained for CD62P and any other markers you may need, then analyzed by flow. The blood should be used within 2 hours of collection.
There are two things I should caution you about- first, do not attempt to isolate platelets prior to staining.This will undoubtably cause large scale isolation. Second, you will need to determine a positive control, as at no time will you find that there is no basal expression of CD62p in a blood sample (TF from the site of punter will activate some platelets). You can do this by treating a parallel sample of blood with ADP for 5 minutes prior to staining. You can use an isotope control or unstained sample to determine negative, and the ADP treated sample to determine positive for gating purposes.
I would suggest reading
Middelburg RA, Roest M, Ham J, Coccoris M, Zwaginga JJ, van der Meer PF.
Transfusion. 2013 Aug;53(8):1780-7
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