Isolation kits

Written informed consents approved by UCLA Institutional Review Board (IRB) were obtained from healthy blood donors and all the procedures were approved by the UCLA-IRB. Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated as described before [33 (link)]. Briefly, PBMCs were obtained after Ficoll-hypaque centrifugation. PBMCs were added to the tissue culture plates, after which the adherent sub-populations of PBMCs were detached and the monocytes were negatively purified using isolation kits obtained from Stem Cell Technologies (Vancouver, Canada). Murine monocytes were also negatively isolated from bone marrow cells. Greater than 95% purity was achieved for each subset based on flow cytometric analysis of CD14. Purified monocytes were cultured in alpha-MEM medium containing M-CSF (25 ng/mL) and RANKL (25 ng/mL) for 21 days, or otherwise specified. Medium was refreshed every 3 days with fresh alpha-MEM containing M-CSF and RANKL. Purified monocytes were cultured in RPMI medium containing GM-CSF (150 ng/mL) and IL-4 (50 ng/mL) for 7 days to generate dendritic cells.

Fresh human peripheral blood leukocytes from plateletpheresis were obtained from Innovative Blood Resources (St. Paul, MN). PBMCs were further enriched by Ficoll-Paque Plus (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) gradient and then NK cells were purified by negative depletion using isolation kits from StemCell Technologies (Cambridge, MA) or Miltenyi Biotec (Auburn, CA), as per the manufacturer’s instructions, with > 95% viability and ≥ 90% enrichment of CD56⁺ CD3⁻ lymphocytes. Viable cell counting was performed using a Countess II automated cell counter (Life Technologies Corporation, Bothell, WA).