Antibiotic antimycotic

To generate iMGs from patients and controls, peripheral blood mononuclear cells (PBMCs) from whole blood were used to differentiate monocytes into microglia-like cells according to a previously published method [25 (link)]. Briefly, PBMCs were isolated by density gradient centrifugation using Ficoll (GE Healthcare, Uppsala, Sweden) and resuspended in RPMI-1640 (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% antibiotic/antimycotic (Invitrogen, Carlsbad, CA, USA) and incubated at 37 °C overnight with 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, Minneapolis, MN, USA), and recombinant interleukin (IL)-34 (IL-34) (R&D Systems) for 14 days to cultivate iMGs cells. To generate fibroblasts from identical FTD–GRN patient, adult human fibroblasts were extracted from forearm skin by punch biopsy and cultured at 37 °C with 5% CO₂ in Dulbecco’s modified Eagles’ medium (DMEM) supplemented with non-essential amino acids (Gibco), sodium bicarbonate (Sigma-Aldrich), 1% (vol/vol) penicillin/streptomycin/Fungizone (Cellgro), and 20% FBS.

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.