One trimacone will generally provide 500 million - 2 billion PBMCs.
NK cells are generally 5% of PBMCs (ranging from about <1% to 30%), so 100 million PBMCs will routinely yield 5 million NK cells. Be aware that the lower the percentage of NK cells, the lower the post-enrichment purity with a negative selection kit. This may negatively impact later assays if the percentage of NK cells amongst PBMC was <1%. Routine post-enrichment purity checking by flow cytometry with a live/dead marker, anti-CD56 and anti-CD3 antibodies, is encouraged
Blood products carry the risk of blood borne pathogens. Follow your local safety guidance.
If needed, NK cells can be cultured and activated with cytokines for enhanced function. Increasing function can help differentiate between experimental conditions, but does change NK cell behavior compared to endogenous function.
1. Base media is R10: RPMI (Gibco Cat. No. 2240-089) + 10% fetal bovine serum (Gibco Cat. No. 26140079) + 100 U/mL Penicillin and Streptomycin (Gibco Cat. No. 15140122).
2. NK cells like to be at high density, we recommend 4x106/mL in flat bottomed TC-treated plastic
e.g. 4x106 cells in 1 mL in a 24 well plate well. Or 1.5x106/mL in 200 μL U-bottom 96-well plate wells.
3. 1 ng/mL IL-15 will enhance survival, but will not overly activate them or drive excessive proliferation (although we find batches from different manufacturers have differing potency, so check your batch with a proliferation assay). 4. 10 ng/mL IL-15 or 100 U/mL IL-2 will strongly activate the NK cells driving enhanced cytotoxicity after overnight treatment and enhanced proliferation of 7 days.