Any better reagent for isolation of PBMC from whole blood? - (May/14/2008 )
Hi. I am trying to find a better reagent for the isolation of PBMC from whole blood. I am currently using Ficoll-paque PREMIUM, which is very frustrating as the PBMC gets contaminated with red blood cells. Can anyone help me with this? Thank you.
Sucrose gradient, see third result:
Are you drawing blood yourself? You clould look at a product from Becton & Dickinson called the Vaccutainer CPT. Basically a glass tube with an 8 mL draw capacity. Has a Ficoll gradient already made up. You just draw the blood into the tube and spin. I haven't had any troubles with it. Costs about $10/tube and you get approximately 1.3 x 106 cells / mL whole blood.
Thank you so much crashy and cellcounter. will try it out. Thanks for your suggestions.
I use the ficoll paque and the accuspin tube (sigma-aldrich) and get no hassle results. Just add 15 mL of the ficoll to the tube, spin for 10-15 sec so the ficoll is under the membrane, add the blood (jus pour it no dilution, no adding drop by dropl) centrifuge 15-20 min and voila you will have the red cell under the membrane and over the membrane the ficoll, buffy coat and plasma beautifully separete. The accuspin are a little bit costly but if need to do a lot of sample you do it more fast and with a good separation for me it worth every penny because I do lots of samples per week.
It sounds easy and effective. But, cost is a factor most of the times.
We are doing pretty fine without any problem with density gradient with Ficoll.
Points I would consider are the dilution of blood, ration of Ficoll and blood, and the temperature of Ficoll-paque.
Dilute the blood in PBS 1X (sterile) and add to the ficoll very very slow and that the liquid runs over the walls of the tube(not directly over the ficoll) so they don't mix then centrifuge at least 20 min (2,200 rpm)
temperature of the Ficoll should be around 20 degrees
Thanks alot for the tips. I would love to use the accuspin tubes but cost is a problem for me. anyway, thanks alot everyone. really appreciate it.
I am new in this forum,
I am working on the lymphoblasts cells.I derived them from the peripheral blood of the acute lymphoblastic leukemia patients using the same methad(Ficoll)
I have a little bit RBC contamination too but my big problem is that my cells slow down to grow and become smaller
Is there any growth factor that I should use?
could you please help me?I really need it
Thanks in advance