Easysep selection kits

Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors (Virginia Blood Services, Richmond, VA) using Sepmate™-50 (Stemcell Technologies) and frozen in 90% FBS/10% Dimethyl Sulfoxide (DMSO). CD45⁺, CD33⁺, or NK cells were purified from cell mixtures using EasySep selection kits (Stemcell Technologies). CD45⁺ cells were purified from co-culture of PBMCs with uninfected/infected Huh7.5.1 cells after 7 days and stained for MDSC markers by flow cytometry. In parallel experiments, CD33⁺ cells were obtained from co-culture of PBMCs and uninfected/infected Huh7.5.1 cells and were subsequently co-cultured for 2 days with autologous NK cells in RPMI1640 containing 10% FBS, penicillin/streptomycin (10μg/mL), and L-glutamine (2mM) at a ratio of 1:2. Purity of autologous NK cells was confirmed via flow cytometry as >82% CD56⁺ cells and <2.5% CD3⁺ cells. NK cells were stimulated with IL-12 (10ng/mL, PeproTech), IL-18 (10ng/mL, R&D Systems), and IL-2 (4μg/mL, eBioscience). The ROS scavenger catalase (100U/mL, Sigma-Aldrich, St. Louis, MO), L NG-monomethyl-L-arginineacetate (500 μM, Sigma-Aldrich), or N(ω)-hydroxy-nor-L-arginine (500μM, Cayman Chemicals, Ann Arbor, MI) was added during the 2-day co-culture of CD33⁺ cells and NK cells.