PBMCs were isolated from buffy coats using Lymphocyte Separation Medium (Corning). Human blood related work has been approved by the Rutgers University Institutional Review Board (IRB). For NK cell expansion, 5 × 106 PBMCs were cultured with 1 × 107 10,000-rad-irradiated feeder cells in 35 mL RPMI 1640 media with 10% FBS (Corning), 2 mM L-glutamine (Corning), 100 U/mL penicillin-streptomycin (Corning), 200 U/mL IL-2 (PeproTech), and 5 ng/mL IL-15 (PeproTech) in G-Rex 6 Multi-well cell culture plates (Wilson Wolf). Media were changed every 3–4 days, and 2 × 107 cells were kept in each well for continued culture at each time. Total cell numbers were counted using trypan blue with an automated cell counter (Nexcelom Bioscience, Lawrence, MA, USA). To determine the percentage of NK cells, cells were stained for CD3 and CD56, followed by flow cytometry analysis.
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