Recombinant granulocyte macrophage colony stimulating factor gm csf

The culture medium consisted of RPMI 1640 and F12 (Gibco-BRL, Life Technologies Corporation, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone, Thermo Fisher Scientific Inc, Waltham, MA, USA). Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 were purchased from R&D (Minneapolis, MN, USA). The inhibitors for NF-κB activation, including Bay11-7082 and PDTC, were purchased from Calbiochem (Merck KGaA, Darmstadt, Germany) and Sigma (St. Louis, MO, USA), respectively. The fluorescent-conjugated antibodies (Abs) recognizing CD80, CD86, CD83 and HLA-DR were purchased from BD Pharmingen (BD Biosciences, CA, USA). Anti-CCR7, recombinant IFN-λ1 and chemoattractants, including CCL19 and CCL21, were purchased from R&D Systems. Anti-Flag antibody was purchased from Sigma. Anti-NS1 antibody was purchased from Genetax (GeneTex Inc, Irvine, CA, USA). Anti-phosphorylated IRF-3 and anti-phosphorylated IRF-7 were purchased from Cell Signaling (Beverly, MA, USA). Antibodies recognizing total IRF-1, total IRF-3, total IRF-7, IFN-λR1, COX-2 and upstream transcription factor2 (USF2) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

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.

The culture medium consisted of RPMI 1640 (Gibco-BRL, Life Technologies Corporation, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum (FBS; Hyclone, Thermo Fisher Scientific Inc, Waltham, MA, USA). Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 were purchased from R&D Systems (Minneapolis, MN, USA). Mitogen-activated protein kinase (MAPK) inhibitors (SB203580 for p38, PD98059 for ERK and SP600125 for JNK) were purchased from Calbiochem (Merck KGaA, Darmstadt, Germany). The antibodies (Abs) recognizing CD80, CD86, CD83 and HLA-DR were purchased from BD Pharmingen (BD Biosciences, Franklin Lakes, NJ, USA). Antibodies recognizing total or phosphorylated c-Jun N-terminal kinase (JNK), p38 and extracellular signal-regulated kinase (ERK) were purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or Cell Signaling (Beverly, MA, USA). Antibodies against p65, p50, p52, RelB, c-Rel, Na⁺/K⁺-ATPase α, Toll-like receptor-3 (TLR-3) and upstream stimulatory factor (USF)-2 were purchased from Santa Cruz. Anti-CCR7, anti-IL-12p40 and anti-Gal-9 Abs and chemoattractants, including CCL19 and CCL21, were purchased from R & D. Anti-Tim-3 neutralizing Abs (LEAF™ purified anti-human Tim-3 antibody) were purchased from BioLegend (San Diego, CA, USA).