A question to you Spectra Optia wizards:
How do you tweak the collection process to get as many lymphocytes as possible from a non mobilized donor while keeping RBC's, granulocytes and possibly also platelets low. We tried collection pref up to 70 and different collection rates, but we have not found the sweet spot of high yield and high purity. 3 to 5E9 would be nice to have, maybe more...
What purification steps do you recommend afterwards? There must be alternatives to ficoll and magnetic beads.
As for purification... yeah... there are no other ways that I know of besides ficoll (or some other gradient) and beads. Especially if you want pure populations. Unless you want to sort by flow, but we only use that for low yield or cells that require multiple markers.
When you say lymphocytes, I'm assuming that you want both Ts and Bs? So basically you need the PBMC layer (through the ficoll, percoll, histopaque, etc) with a positive selection for monocytes.
Thank you, Shen-An. Yes, I am looking for Ts, NKs and Bs. As far as I know there is no way of seperating them mechanically, as their morphology is too similar. I'm fine with PBMC's, but I'm dealing with 1to 2E10 cells, and I need them aseptical and untouched. So flow is not an option here and beads don't seam feasible either.
Even if I collect at HKT 2 or lower, the residual RBCs ant PLTs still outnumber the PBMCs. I can get rid of the PLTs by repeated washing, but it's hard to get the RBC's out with Ficoll, since the RBCs that passed the Optia are the ones that you can't seperate well by ficoll.
Actually... miltenyi does make a system that allows you to separate large numbers of cells. using the beads. We had this system installed in the hood and everything is under aseptic conditions.
Yes.. the beads does get pricy after a while.
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