Penicillin streptomycin fungizone

Adult human fibroblasts were extracted from the forearm skin by punch biopsy. Fibroblasts were cultured at 37% with 5% CO₂ in media containing Dulbecco’s modified Eagles’ medium, non-essential amino acids (Gibco, Grand Island, NY, USA), sodium bicarbonate (Sigma-Aldrich) and 1% (vol/vol) penicillin/streptomycin/fungizone (Cellgro), supplemented with 20% heat-inactivated foetal bovine serum.
For immunostaining, cultured fibroblasts were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature, permeabilized with 0.2% Triton X-100 in PBS for 15 min and blocked with 1% bovine serum albumin in PBS for 1 h. Then, the cells were incubated with mouse anti-G3BP antibody (1:1000, Millipore) overnight at 4% and labelled with fluorescein isothiocyanate-conjugated secondary antibodies (1:200, Jackson ImmunoResearch) for 60 min at room temperature. Images were acquired with a Leica TCS SP8 laser-scanning confocal microscope (Leica) using an HC PL APO CS2 63x/1.40 objective.
To quantify SG dynamics, we counted cells containing more than two puncta of G3BP1-positive granules in a randomly selected region (>50 fibroblasts). The experimental unit in this assay was individual fibroblast cultures. Results represent three independent fibroblast cultures (n = 3).

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.

Fibroblasts were obtained from this patient and healthy controls. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% FBS (Gibco, Carlsbad, CA, USA), nonessential amino acids (Gibco), sodium bicarbonate (Sigma‐Aldrich, St. Louis, MO, USA), and 1% (vol/vol) penicillin/streptomycin/fungizone (Cellgro, Manassas, VA, USA) in an incubator at 37°C under 5% CO_2.HEK293T cells were cultured in DMEM with 10% FBS in an incubator at 37°C under 5% CO₂. For transient overexpression, cells were transfected with Lipofectamine 2000 (Life Technologies, Grand Island, NY, USA) according to the manufacturer's instructions.