I use human blood samples (~8ml each) from healthy individuals and perform centrifuge over lymphoprep, in order to isolate the PBMCs. Then, I use the EasySep Kit to isolate the CD19+ cells. The purity is supposed to be ~99%, but I get much less (only once managed to achieve a percentage close to 80% of the B cells). As the samples are 8ml of blood from healthy donors, there are fewer cells than 10^8 cells/ml (the indicated cell concentration, according to the manual) and I scale down.. Any ideas/tips??
Thanks
In my experience using magnetic bead separation... while it sounds simple to use a magnet with a tube, you get MUCH better purity using a column (Miltenyi was what I preferred).
I would call StemCell, they should have some tips for you. They have to tell you how they get to 99% purity.
Hi, I would definitely email our tech support ([email protected]) with the description of your experiment and they should be able to help you. Very often if the starting sample is too small, adding the antibody cocktail as per protocol could result in too high concentration of antibodies vs target cells, which can result in non-specific binding, hence lower purity.
The recommended starting concentration of PBMCs is 1x10e8 PBMCs/ml. But you can work with as little as 0.1ml sample volume (if you are using purple magnet) or 0.25ml (if using silver magnet).
Anyhow, please email our tech support with your situation and they will recommend you what would be the best way to improve your results. I am confident you will get the better purity than 80%.
Alternatively, you can try EasySep Direct B cell isolation kit (Cat #19674), which will allow you to isolate B cells directly from Whole Blood without the need of PBMCs isolation (so not loosing as much cells during the density spin and wash step). You can request a sample of this kit to try. The protocol takes no longer than 30 mins from whole blood to isolated B cells.
Best of luck!
I too have heard about better purity using columns from Miltenyi, but there's definitely a way for the EasySep kit to work. I'll keep trying and also contact the tech support.
Thank you both for your reply!
- Badges
- Science method