The NK cell culture method I chose is to add K562 and allogeneic PBMCs to the system, which have been treated with 100ug/ml MMC. Freshly isolated PBMCs are sorted by EasySep™ Human NK Cell Isolation Kit to obtain NK cells, and then added to feeder cells. Each 96 U-shaped well is added with 20,000 NK, 30,000 K562, and 150,000 allogeneic PBMCs. NK medium components include RPMI 1640 mixed with MEM Non Essential Amino Acid Solution, sodium pyruvate, FBS, L-Glutamine and P/S, rhIL-2, rhIL-15, PHA-M, and human AB serum. Now, I have encountered the following trouble:
1. At the 14d of culture, there are more T cells being expanded. This may come from the remaining T cells after magnetic bead sorting or the allogeneic PBMCs not been treated with MMC well? How to effectively solve this problem?
2. How long is it better to pass or add culture medium? How often should the feeder cells be replaced?
3. Which method of lentivirus infection, retrovirus infection and electrotransduction is better for gene transduction of NK cells?
Thanks for your answers.
Fei Gao The same question n topic has been discussed on this link
researchgate.net/topic/Natural-Killer-Cells
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