Isolation and Expansion of Human NK Cells

Natural killer (NK) cells were prepared from freshly collected plasma apheresis peripheral blood obtained from healthy donors at the Life Stream Blood Bank (San Bernardino, CA, USA), in accordance with Loma Linda University IRB protocols. Peripheral blood was prepared as described previously [47 (link)]. NK cells were obtained from the total lymphocyte fraction by negative immunomagnetic cell separation using either EasySep or RosetteSep negative selection kit (STEMCELL, Vancouver, BC, USA) or MACS magnetic NK cell isolation kit (Miltenyi Biotec, Auburn, CA, USA). The purity of the NK cell population was determined by flow cytometric analysis and ranged from 85–95%, with all kits showing comparable performance in obtaining CD3-/CD56+ NK cells (greater than 90% purity and viability). NK cells were cultured at a high density of 5 × 10⁶ cells/well in 6-well non-pyrogenic polystyrene culture plates overnight in RPMI 1640 media (Mediatech Inc. Manassas, VA, USA), supplemented with 10% FBS (Hyclone, Logan, UT, USA), 1 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 100 U/mL human recombinant IL-2 before use in experiments.