I isolate PBMCs from leukocyte reduction filters derived from healthy donors. The filters are purchased from Rhode Island Blood Center. I use HBSS buffer with 2.5% surcose and 5mM EDTA (PH=7.2) to back flush the blood, and use Ficoll to isolate PBMCs. I only get 100 millions cells. In theory, one filter can generate up to 500 million cells.
Do you take the filter from a blood donation of 500ml? Does your literature, which reports a five-fold amount of leucocytes do that as well or do they use a filter taken from another procedure like an apheresis? Is their reported number of cells also taken from healthy donors?
Like Erin, I suspect the ficoll-step, too.
Thank you for following, both.
Erin, I think I collect the whole layer. The layer actually looks pretty thick. Eva, I am not sure the filter comes from what kind of procedures in the hospital. Will that affect the cell yield? Can you explain more about that? The reported number of cells are also taken from healthy donors.
Actually, in the most recent experiment, I count the cell number right after I collect the whole layer, it is 200million cells. But after washing twice by using DPBS without EDTA, I lose half of the cells. I know washing procedures will lose cells, but I it will make us to lose so many!
Perhaps you can compare your procedure to the one from literature a bit more in detail? Do they also report the number after washing?
I could well imagine, that a filter used for an apheresis procedure (like e.g. plasmapheresis or platelet apheresis) collects more cells, as the blood is proceeded several times.
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