I'm working with lymphocyte isolation from 10ml whole blood using lmyphoprep isolation media but didn't get the clear buffy layer. Which most likely the cause?
1. I just noted that the vacuum lithium heparin blood collection tube was expired (Dec2016)
2. The centrifuge speed,according to the protocol the speed should be at 800g,at room temp 20' and for 20min, but i made it at 1200rpm, as i got problem to convert the rpm to g
3. The protocol said to use nacl 0.9% to dilute the blood but i used PBS solution for dilution.
4. Any other possible cause?
Your expert comments/responses ia highly appreciated. Thank you very much
@ummu,
Specifically 1 & 2. But I will go with 2 more. So, be careful next time.
BR,
Babu
Unless the whole blood is coagulating, the expired tubes are fine.
Ok... since this is a gradient, I'm going to assume that you are layering the whole blood correctly? And you aren't "mixing" the whole blood into the lymphoprep?
What did your tubes look like when you spun at 1200rpm? THis may no may not be sufficient depending on the rotor size. But usually, 1200rpm is NOT even close to 800gs... it's usually about 400 - 500 or so. I would do better calculations for the conversion if your centrifuge does not have a RCF setting.
I agree with Shen-An Hwang. If your blood didn't clot, then it is not your blood collection tubes. When working with blood, we usually dilute them in PBS and we have never run into issues. Hope you did carefully layer the diluted blood on top of the lymphoprep with minimal mixing. Your conversion might have been the problem. The tubes need to be centrifuged at the recommended speed for 15-30 mins with the brake OFF. If they get disturbed even a bit or if you use the wrong settings, this would have a major impact on the separation.
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