Generating Myeloid Adherent Cells from hPBMC

Human blood was drawn from healthy donors following the guidelines of the Institutional Ethics Committee, NISER, Bhubaneswar (NISER/IEC/2022-04). The procedure for generating myeloid adherent cells from human peripheral blood mononuclear cells (hPBMC) was followed as described elsewhere with little modifications (38 (link)–41 (link)). Briefly, circulating monocytes were enriched by 2 h adherence after Hi-Sep LSM (catalog number: HiSep LSM™ 1077‐ LS001; HiMedia Laboratories Pvt Ltd, India) based density gradient-centrifugation according to the manufacturer’s instructions. The adherent cells were cultured in RPMI-1640 supplemented with 10% FBS, antibiotic-antimycotic solution and L-glutamine for 3–5 days. The adherent cells obtained after 96 h were of monocyte-macrophage lineages (more than 97%) as found enriched with CD14⁺CD11b⁺ population (42 , 43 (link)). The monocyte-macrophage cells derived from hPBMC were seeded in 12 well plates (Thermo Fischer, USA) at a density of 0.8x10⁶ cells/well. After 24 h of seeding, pre-incubation was carried out for 3 h with 1 µM of TAK-242, followed by CHIKV infection with MOI 5 for 2 h (19 (link)). The infected cells were harvested at 8 hours post-infection (hpi) and downstream experiments were conducted.