I am testing some compounds effect on platelet glycoproteins. Could anyone suggest me what protocol I should use especially by using flow cytometry? Can I use 2-3 days old platelet for the assay?
The best approach (if using human platelets) is PAC-1 from BD. They are the only company that sells a GPIIbIIIa antibody that detects the active form of the complex. We usually use fresh platelets, which if fixed can be stored for up to 2 days prior to analysis by flow cytometry. I have never used 2-3 day old platelets, so am not sure about the activity/reproducibility of integrin activation under those conditions. If you are using mouse platelets, you will need to use JON/A antibody instead of PAC-1.
The best approach (if using human platelets) is PAC-1 from BD. They are the only company that sells a GPIIbIIIa antibody that detects the active form of the complex. We usually use fresh platelets, which if fixed can be stored for up to 2 days prior to analysis by flow cytometry. I have never used 2-3 day old platelets, so am not sure about the activity/reproducibility of integrin activation under those conditions. If you are using mouse platelets, you will need to use JON/A antibody instead of PAC-1.
Hello,
the second question is simple to answer:
No, you should always use freshly drawn blood and process the sample right away, i.e. within 20 min to get comparable results.
As for the best protocol, that depends on the marker you wish to test, the flow cytometer you have and so on. Our group published two paper in 2011 with more detailed methods.
Ostertag, 2011 Mol Nutr Food Res, In vitro anti-platelet effects of simple plant-derived phenolic compounds are only found at high, non-physiological concentrations
and
de Roos, 2011 Eur J Nutr 50:553–562, Anti-platelet effects of olive oil extract: in vitro functional and proteomic studies
I hope that helps to get you started.
Kind regards
Eva
Fresh platelets should always be used. In my experience, storing platelets dramatically changes the level of transmembrane proteins. If you are assessing the surface expression of GPIIb/IIIa then the anti-CD41 and CD61 antibodies from BD are great. If you want to assess activation of GPIIb/IIIa, then you will require PAC-1 from BD. I have used fixed platelets in some assays for detecting GPIIb/IIIa and while there is some loss of signal it is an option. However, the product insert for the GPIIb/IIIa antibodies from BD discourage fixing the platelets prior to stainng. As already stated by others, these antibodies are for use with human platelets. There are however reagents for mouse platelet work.
From my experience storing influence both the percentage of GPIIbIIIa positive platelets and the density of this activation marker. The best way is to follow one of standardized protocols defined in:
Immunophenotypic analysis of platelets.
Krueger LA, Barnard MR, Frelinger AL 3rd, Furman MI, Michelson AD.
Curr Protoc Cytom. 2002 Feb;Chapter 6:Unit 6.10.
This paper is good because it defines all other things relevant to the analysis, blood sampling (to prevent extracorporal activation), anticoagulants to be used, fixing procedures. Also useful because it shows how you should gate the platelets ect.
I usually use CD61-PerCP (BD), Pac-1-FITC (BD) and CD62P-PE (BD). PAC-1 is only available labelled with FITC, so you need to use any of available panplatelet markers (CD61 is not the only one) labelled with any other fluorescent dye.
I would suggest you to use an activator such as TRAP, ADP or as more recently used some activating peptides as positive control. Current Protocols in Cytometry chapter on platelets is certainly a good start for protocols. Two volume of Methods in Cell Biology dedicated to cytometry (edited by Z. Darzynkiewics) also have a good chapter on platelet analysis.
It would be easier to reply if you specify what species, whether in vitro or ex vivo, what you mean by "effect on glycoproteins", etc...
Thank you very much for the responses. We are using human blood to test some natural product effect on platelet aggregation in vitro. However, at the moment, we have not had the ethical approval to work on fresh blood yet.
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