Hello everyone,
I'm trying to find a way to isolate PBMC from very small blood samples (approx. 250µL, but sometimes less). I already tried using a RBC lysis buffer, centrifugation and washing, but I lost too much material. So I would like to try with Ficoll or Leucosep techniques, but it's recommanded to use larger amount of initial sample (2mL).
In the instructions for Ficoll-Paque PLUS or Ficoll-PAQUE Premium (GE Healthcare), they say that smaller blood samples can be managed by modifying the recommanded procedure, but there is no more informations than that.
Does anyone already try Ficoll or Leucosep (or other) from very small blood samples ? What are the required modifications of the original protocol ?
Thanks !
I already did that on samples 24h culture show coagulation and it becomes messy to harvest anything).
Good luck!
What Ricardo says is true. If you still want to give a try is follow the recommended protocol in scale, like instead of using 15 ml tubes use 2 ml microtubes. another thing that may help is to dilute the sample a little further than the recommended (1:1 sample:PBS) to expand your initial volume. For this try not to exceed 1:3 dilution.
I have never tried this before but at least this is how I would do it.
Good luck
I already did that on samples 24h culture show coagulation and it becomes messy to harvest anything).
Good luck!
First tell us what do you want to do with your PBMC after isolation?
Thank you all for your answers.
Actually, I'm working on a virus and I'm trying to isolate viral RNA from blood samples. My point is to discover if the virus is circulating in the blood or in the blood cells. So I don't need to have a lot of PBMC, neither have alive PBMC, because then, I will perform a qRT-PCR on the PBMC isolates and theoretically, a very small amount of genetic material is sufficient and can be amplified.
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