Given a buffy coat that was formed from the centrifugation of whole blood, what is the best way to isolate the WBCs, since the buffy coat contains both WBCs and platelets? I would like to inspect an isolated sample of just WBCs under the microscope.
Note: I am interested in all types of WBCs, including neutrophils, basophils, and eosinophils, monocytes and lymphocytes. Multiple protocols that can isolate different subpopulations are fine, as long as we can eventually inspect all types of WBCs.
I would add a step of ACK lysis to lyse any remaining RBCs. I've never tried to remove platelets, I've never found them to obscure any other WBCs. Too much manipulation of your sample and you risk eliminating other WBCs and skewing your results.
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You are correct that buffy coat contains lots of platelets (nearly 50 times more than WBC). One way you could reduce (but cannot totally eliminate) platelet contamination is by first spinning at low speed (1000rpm) to bring down the RBC and WBC. Remove the platelet rich plasma and resuspend the cells in saline and centrifuge again to prepare the buffy coat. If you really want to prepare a 'pure' population of WBC, you will need to use magnetic beads with an antibody against platelet specific antigen like CD62.
Good luck
Siva
Hi Dennis
Further to Siva's suggestion of low speed centrifugation, if platelets continue to be a problem, you could repeat this step by resuspending the WBCs & RBCs in saline, centrifuge again at low speed, carefully remove the top two-thirds of the platelet-rich-saline and then continue as Siva suggested with the buffycoat centrifugation. Note that granulocytes are heavier than mononuclear cells and will tend to enter the RBC fraction, so you need to be mindful of this if you want to collect all WBC types.
Regards, Rosemary
Specific gravity of cells is one way to do it using centrifugation or solutions that layer in between the cell types based on the above. See attached figure. I drew this and it is copyrighted, but you have my permission to use it..
The other method is using a cell sorter, and you will have to read up on that. These are expensive but large research institutions have them (US $300K).
As an aside, granular leukocytes have a tendency to degranulate easily. Nevertheless we use granulocyte transfusions to treat patients who have gotten a bone marrow transplant and are septic. Granulocytes are collected using tri-sodium-citrate + hyydroxyethylstarch "Hespan".
What do you want to do with the cells?
This is an interesting thread...reached here looking for ways to minimize platelets from buffy coat derived PBMCs to use in single cell sequencing. will try differential centrifugation and cd62 bead to see what works best. An additional issue i have is that am trying to use frozen stocks of PBMC (in DMSO)- dont know how freeze thaw impacts platelets. Thanks for any inputs..
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