I have been isolating platelets and PMBCs using BD vacutainer CPT tubes. After the first spin, RBCs are removed underneath the gel layer, and PMBCS and platelets form a monolayer. I then separate the PMBCS and platelets by spinning @ 300g to pellet PBMCs, and aspirate off the supernatant containing platelets.
My question is: If I then pellet the platelets with a faster spin (800g is what I have found from other protocols), will they now be activated? We lyse them very soon after, so maybe it does not matter very much. But still, I would like to know if anyone has an answer to this?
Thanks very much for any help!
The platelets will be activated with every centrifugation. If you aim to have unactivated platelets, blood collection should be made in to heparinized tubes (blue top BD tubes). If you do immunophenotyping for CD62p (activation marker for platelets) or CD63 you will see that their expression have increased in your samples.
Thank you Gulderen for your answer. Do you think it is still worthwhile to examine the activated platelets? I am new to this field but I assume unactivated platelets would be ideal. But, I also assume we should still be able to pull out some differences between different cohorts of subjects. What do you think?
Thanks again.
This depends on what you are going to study with them. If the activation will effect your results, I can provide you a whole blood protocol but then you will need to use a fixative solution (PFA). Also 800 g is very high centrifugation speed for platelets.
Ok - what measurements do you do after fixing? Is this for flow cytometry or something different?
After fixing you can make immunophenotyping by using cytometry.
Every time the centrifuge is used, the platelets will become active. Blood should be collected in heparinized tubes if you want to obtain inactivated platelets.
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