Hello dear collegues! Im using gradient centrifugation for isoltation of PBMC from blood samples.
5ml of fresh blood + 5ml of ficoll 1077 at 400g for 30min, 18C.
I remove plasma layer and collect 2ml of upper ficoll layer with PBMC. Then i add 10ml of PBS+BSA(10g/L) solution, mix and centrifuge at 100g for 20min, then remove supernatant. I repeat washing step 3 times but still have a lot of platelets (pic). Is there any simple method to remove platelets completely?
The weird thing is most platelets should be above your PBMC layer, not below it. Which means that they should have been in the plasma layer, or most of them anyway. (And I'm going to assume that you didn't have the break on on your centrifuge when spinning down the ficoll and blood?)
When you isolate your PBMCs, you do see a white band between the plasma and the ficoll right? I would focus on taking this white layer, then just 2mL of the ficoll layer.
In my hands, despite collecting the PBMCs layer, there is always some RBCs left over, so I alwyas recommended a RBC lysis step.
My suggestion is: After you collect the PMBCs, put this into a 50mL tube. Add 1xPBS up to 50mL, and spin at 300 to 400gs for 10 minutes (with the break). Lyse the RBCs. And wash with 10mL of 1xPBS 2 or 3 times.
Hi
I think this paper is help you:
Cazenave JP, Ohlmann P, Cassel D, Eckly A, Hechler B, Gachet C. Preparation of washed platelet suspensions from human and rodent blood. InPlatelets and megakaryocytes 2004 (pp. 13-28). Humana Press.
Follow this link
https://www.ncbi.nlm.nih.gov/m/pubmed/22936376/
regards
this link is useful
https://www.ncbi.nlm.nih.gov/pubmed/22936376
regards
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