Cryopreserved human pbmcs

Cryopreserved human PBMCs were purchased from Cellular Technology Limited. Human NK cells were isolated from PBMCs using Miltenyi NK Cell Isolation Kit (Miltenyi #130-092-657) with MS Columns (Miltenyi #130-042-201), and a MiniMACS Separator (Miltenyi #130-042-102). Stimulation of primary human PBMC and NK cell fractions were performed as described above.

Cryopreserved human PBMCs were purchased from Cellular Technology Limited (USA). The cells were thawed in accordance with the manufacturer’s instructions and seeded at 2×10⁴ cells/well onto 384-well cell culture plates in RPMI1640 medium containing 10% FBS and compounds. Four hours later, the cells were stimulated with 20 ng/ml LPS for 4 hours and 2 mM ATP (Sigma-Aldrich) for an additional hour. The relative cytokine levels were measured using the HTRF Kit as described above.

1,2-Distearoyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG-2000-Biotin), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG-2000-Amine) were purchased from Avanti Polar Lipids (Alabaster, AL). IL-2, IL-6, and TNF-α ELISA kits were purchased from BioLegend (San Diego, CA). Cryopreserved human PBMCs were purchased from Cellular Technology Ltd (Cleveland, OH). FITC-avidin was purchased from Thermo Fisher Scientific (Waltham, MA). Dulbecco's phosphate-buffered saline (DPBS), chloroform, LPS, and MPA were purchased from Sigma Aldrich (St. Louis, MO). The anti-HIF-1ɑ antibody was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX).