Hello,
I am trying isolation of platelets from human and rat blood. I have collected different protocols and one thing is common in all is the use of four different buffer that are ACD, CPD, wash buffer and HEP buffer.
Second way is the use of ficoll directly to get platelets rich plasma, washed, and use for the experiments.
How these two method are different?
Regards
Dear Sumeet,
I hope that the following information will be helpful.
http://www.abcam.com/protocols/isolation-of-human-platelets-from-whole-blood
Hi Sumeet,
Please find attached a very common protocol used for platelet isolation using centrifugation. Major differences are 1) citrate is used instead of ACD or heparin as the anticoagulant, 2) prostaglandin I2 is used to keep platelets resting and 3) vacutainers are not used to prevent shear-induced platelet issues.
Good luck,
Hey Sumeet,
Depending on how you plan to use the platelets you should adjust your protocol accordingly. For washed platelets the above protocol is as good as any. Fickle is a great technique for lymphocyte isolation but sub-optimal for platelets. You must remember that any time you wash platelets you activate them to a degree, and this results in artifactual errors in many applications. It is alway best to use whole blood when possible for Flow Cytometry, aggregometry, and function testing. Some protocols, such as the CAT require PRP- but the further you get away from whole blood after PRP the further you get from a true physiological response. This is why newer tests such as the platelet mapping assay for the TEG have found ways to define functional deficiencies in platelets that are not related to coagulation protein function in whole blood.
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