Does anybody have experience regarding immunomagnetic isolation of NK cells from human buffy coats(cca 1x10e9 PBMCs)? What is the maximum number of cells one can obtain by microbead-selection?
Tnx!
I have attached a link that I used to refresh myself on the percentages. Probably 10% of the PBMCs in your buffy coat would be NK cells. It can vary from person to person. I would do three selections: first, plate in shallow medium in a cell culture dish for an hour at 37C, and gently take off the supernatant (incubating will cause the monocytes to adhere). Spin down the supernatant and do two magnetic selections, a negative selection first to get rid of T and B, and then a positive selection like CD56 to isolate NK. Then sort on a flow cytometer if you need additional purity. You can expect some loss, but I usually recovered about 50% of the available NKs in a given population that way, if I remember correctly. You can magnetically select using fluorescent antibodies, then use the fluorescence on the flow sorter to gate out the NK/T cells (CD56+/CD3+).
http://technical.sanguinebio.com/types-of-immune-cells-present-in-human-pbmc/
From our experience the range of NK cells within PBMC is about 5-15% (analyzed after FiColl). Don´t expect to much. I can also recommend magnetic isolation of "untouched" NK cells (negative isolation). The loss during magnetic seperation varies from 20-50%.
Dear Urban,
In our lab we did some extensive tests to measure the efficiency of the human NK cell isolation from Buffy Coat. In our hands using Miltenyi NK cell isolation kit (isolates untouched NK cells, discarding the rest of the PBMC) we obtain around 98% purity and 50-70% of recovery. That is from 1 x 10E9 PBMC with 10% NK cells one may get around 60x10E6 NK cells. I hope this helps. Semih
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