I'm culturing murine NK cells in vitro. However, the second day post isolation, 80% or more of the cells are dead or apoptotic. The medium I used was a-MEM supplemented with 10% FBS, 10 ng/ml IL-15, 10-7 M Hydrocortisone, 50 μM beta-mercaptoethanol and 1% Strept-Pen. Is this culturing medium suitable for NK growth? I had done 7-ADD staining after NK isolation and the viabiility was above 90%. I wonder whether the culturing medium is too barren to maintain a NK cell. Can anyone explain it and give me some advice?
Hi. The research group I work in have used a simple RPMI-1640 medium +10%FCS, 2mM L-Glutamine, Penicillin (100U/mL)/Streptomycin (100µg/mL), 50µM beta-mercaptoethanol and a mix of Non essential amino acids. We culture with 100U/mL IL-2 but also have a protocol with 10U/mL IL-15. When we use enrichment instead of positive isolation we have viability above 90% after 7-10 days in culture.
It is also worth checking the CO2 calibration of your incubator and the humidity if cells are dead already next day. (Empty humidity pans in incubators are quite deadly for cells.)
Regards,
Kristin
Hi,
You may try CellGro medium from Cellgenix. It works very well with human NK cells for long term culturing. In addition, try to do a negative selection instead of positive selection for NK cells.
Regards
HG
Hi. The research group I work in have used a simple RPMI-1640 medium +10%FCS, 2mM L-Glutamine, Penicillin (100U/mL)/Streptomycin (100µg/mL), 50µM beta-mercaptoethanol and a mix of Non essential amino acids. We culture with 100U/mL IL-2 but also have a protocol with 10U/mL IL-15. When we use enrichment instead of positive isolation we have viability above 90% after 7-10 days in culture.
It is also worth checking the CO2 calibration of your incubator and the humidity if cells are dead already next day. (Empty humidity pans in incubators are quite deadly for cells.)
Regards,
Kristin
Hello,
I'm also interested in that question: I have NK cells from the ATCC but I don't know what they are happy in. We tried the aMEM with beta-mercaptoethanol, Sodium bicarbonate, Folic Acid, Horse serum and IL-2 like the ATCC recommands but they don't seem to proliferate very fast. So I'm adding some questions here:
Do they agglomerate like B cell do?
Do they like medium acidic or does it have to be changed every 2 days?
What is their morphology like?
What is their expected proliferation rate?
Thanks for helping,
Sylviane
Hello,
I am interested in growing and challenging horse NK cells, I need them to ve alive for at least 6 days and then co-culture with other type of cells. Any suggestion for media and selection (positive/negative) protocols?. Thanks in advance
Do NK cells in culture need to be trypsinized for passage or are they floating?
Is 4 hours the minimin hours that NK cells need to be cultured for before harvesting? And do they die after 16 hours. Most of the cell assays I am referring to seems to use these two time points. Can anybody please explain, clarify on why these two time points are chosen. Thanks
I use the similar protocol of RPMI-1640 medium +10%FBS, 1% L-Glutamine, Penicillin (100U/mL)/Streptomycin (100µg/mL), 55µM beta-mercaptoethanol + 10 ng/ml IL-15 (R&D) to culture the murine NK cells in vitro and it seems not to work after 24 hrs of culture though. Does anyone know what might be the reason? Thanks
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