We are attempting to isolate PBMCs from human blood and I have a few questions. We have decided to use leucosep tubes as we will be isolating these PBMCs from patient samples that will arrive at different times of the day and therefore be handled by different people (Leucosep seems to be the most standardized method). Today I isolated PBMCs from 2 members of the lab with different results. One sample gave about 6 x 105 cells/ml and the other about 4.5 x105 cells/ml, which as I understand are quite low. One thing I did notice is that the supernatant after the first wash was still very cloudy, suggesting that maybe there are still cells that were not spun down. When collecting the PBMCs I tried to take as little of the plasma layer as possible but I'm wondering if I had taken too much of the ficoll (3ml max) then this may stop the cells being spun down in the first wash?. Or is this cloudiness just lots of platelets?
All steps are carried out at RT and the blood is diluted 1 in 2 in Mg/Ca free pbs before being added to the leucosep tube.Wash steps are performed at 1200rpm (250g) for 7 minutes.
Any help would be greatly appreciated.