Preparation of peripheral blood mononuclear cells
Posted 18 January 2010 - 01:10 PM
Most of the methods start off: "dilute the blood 1:1 with PBS and layer on top of an equal volume of Lymphoprep/Histopaque 1077...", which means I can't use the plasma. Some say you can layer whole undiluted blood on Lymphoprep, but I want to avoid any contamination of the sample with Lymphoprep, having sensitive assays to do on the red cells and plasma that might be affected.
If I centrifuge the blood sample first (1200 x g for 15 min), the packed leukocyte layer (buffy coat) can be removed and resuspended in PBS-EDTA and layered onto Lymphoprep. However, after centrifugation, the cells form clumps instead of a nice homogeneous band at the density interface, and this is probably due to platelet activation during the initial centrifugation. I could add platelet activation inhibitors to the blood, but I prefer not to be adding things whose impact on the other cells might be undesirable.
A milder initial centrifugation (200 x g) is the established way of producing buffy coat that isn't activated, but then I lose a lot of plasma.
Surely there must be other people out there who are trying to prepare plasma and cells from limited samples and with similar constraints? Am I missing some simple method kept secret by hematologists, or is this really such a difficult problem to solve? If anyone has a simple (and preferably inexpensive) way of solving this problem, I would be very grateful.
Posted 20 May 2010 - 05:02 AM
Posted 20 May 2010 - 06:50 AM
To just get the serum and cells separated, we just use a Ficoll gradient and centrifuge for 30 mins. The result isn't the cleanest prep of PBMCs, but it isn't all that bad.
After that, we use the expensive magnetic beads to ensure a clean population.
Not sure if that entirely answers your question, but hopefully it helps!
Posted 20 May 2010 - 07:08 AM