Rpmi 1640 plus l glutamine

Human monocyte-derived macrophages (hMDMs) were derived from human blood acquired via venipuncture from healthy donors following a Ohio State University Institutional Review Board approved protocol. Written informed consent was provided by study participants. The protocol followed published methods (Schlesinger et al., 1990 (link); Hoang et al., 2016 (link)). Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood over a Ficoll cushion (GE Healthcare Bio-Science, Piscataway, NJ, United States). PBMCs were then cultured in sterile screw-cap Teflon wells in RPMI 1640 plus L-glutamine (Gibco-Life Technologies, Grand Island, NY, United States) with 20% autologous human serum at 37°C in a humidified incubator containing 5% CO₂ for 5 days. Teflon wells were chilled on ice to recover the PBMCs, which were then re-suspended in RPMI 1640 with 10% autologous serum. Cells were then allowed to attach in 24-well or 6-well tissue culture plates for 2–3 h at 37°C in a humidified incubator with 5% CO₂. After washing to remove the lymphocytes, hMDM monolayers were seeded at a density of approximately 2.0 × 10⁵ cells/well for 24-well plates for infection studies.