Isolation and Culture of Induced Microglia-like Cells

PBMCs were isolated by density gradient centrifugation using Ficoll (GE Healthcare, Uppsala, Sweden). iMG cells were established using a previously published method [25 (link)]. Briefly, PBMCs were resuspended in RPMI-1640 (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco) and 1% antibiotic/antimycotic (Invitrogen, Carlsbad, CA, USA) and cultured overnight at 37 °C and 5% CO₂. The next day, adherent cells (monocytes) were cultured in RPMI-1640 Glutamax (Gibco) supplemented with 1% antibiotic/antimycotic, recombinant granulocyte–macrophage colony-stimulating factor (GM-CSF) (R&D Systems), and recombinant IL-34 (IL-34) (R&D Systems) to develop iMG cells. After generating iMGs, the cells were labeled with human CD11b-APCVio770 and CD45-phycoerythrin (PE) (Miltenyi Biotec, Gladbach, Germany), and flow cytometry was performed as described previously [32 (link)]. All data were assessed by FACSCanto II flow cytometry (BD Biosciences, Piscataway, NJ, USA) and analyzed by FlowJo software. Gene expression in iMGs was measured using quantitative real‑time polymerase chain reaction (qRT‑PCR) as described previously [32 (link)]. Primer information is shown in Supplemental Table 3.