I have a problem with methodology, because I would like isolate blood cells for qRT-PCR ans Western blood analysis. My questions are:
1. What volume of blood do I need? 5ml will be enough?
2. How can I separate PMBC without losing granulocytes? To further analysis I need two types of cells (PBMC and granulocytes separately).
3. Who knows which metod will be the best?
Thank You very much and I expect any answers.
Małgorzata
Hi Malgorzata,
As for question 2, I would try a CD45 bead selection or cell sort. If you do a cell sort you could use Live/dead, CD45, CD41 (to dump platelets), CD66b&CD15 (neutrophils), FceR1 (basophils), and Siglec8 (eosinophils) after an RBC-lysis and blocking step (1% Human Ab serum). You could sort your populations of interest directly into Trizol or NP-40.
Another option is Ficol-Paque premium (see attachment).
As for question 1, I do know that 5mL is more than enough for qRT-PCR. I have, however, no experience regarding protein yields.
I hope this helps,
Anouk
Thank you very much for this response. I will try according to your answer. Thank you once again
Hi, you should use Ficoll Hypaque gradient. After centrifugation, PBMCs will be at the interphase between plasma and Ficoll. Granulocytes will be placed above erytrocites red pellet.
Hope it helps!
Thank you very much for answer. I'm worried about efficiency of the ficol gradient because I would like to isolate mRNA and performe Western blot analysis.
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