When i am isolating pbmcs, iam getting a white clot (aggregated PBMCs) in the buffy coat, so I can see just a few number of separated pbmc cells, and this problem conflicts with counting of pbmc cells and as a result I don't know howmany pbmc cells added in each well exactly.
I tried to disaggregate these clots by pipetting many time but that is not fully successful and some clots are being seen.
Hi,
Please check if your PBS is free from Ca+ and Mg+, which could activate the sticky platelets. Also the media should be non-complemented. If it is so, you could try giving an extra wash at low speed to get rid of platelets if any . Usually that is the reason for having aggregated PBMCs .
Hope this helps!
Hello Salih Jabbar
Are you isolating PBMCs from fresh blood? Sometimes DNA is released from dead cells and the sticky nature of DNA cause cells and other debris to aggregate. PBMCs must be isolated from fresh blood within 4 to 8 hours after collection of blood.
Best.
I take it you are using cells from human buffy coat. Was there any anticoagulant involved when drawing blood? If you used buffy coat from blood donations, you may want to have a look at the treatment before you got the material. As mentioned above, cell degradation and platelet activation can cause this kind of problem.
Neha rana, Malcolm Nobre, and G. Weckmann, thank you so much. I solved the problem by using heparin instead of the EDTA during blood collection..
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