Perfused brains were minced with scalpel blades and transferred to C-tubes (Miltenyi Biotech) containing an enzymatic digestion mix consisting of DMEM/F12 (Thermo Fisher Scientific) supplemented with 1 mg/ml papain (#P, 2 U/ml Dispase II, and 20 U/ml DNase I (all from Sigma). Tissue was homogenized at 37 °C using a gentleMACS™ Octo Dissociator with Heaters (Miltenyi Biotech). Myelin debris was removed by density centrifugation of the homogenate over a discontinuous 70%-37%-30% Percoll gradient. Microglial cells were collected from the 70/37-interphase, resuspended in Microglia Culture Medium (MCM, consisting of DMEM/F12+1% Pen/Strep/Glutamine, 10% FCS) and seeded onto 12-well plates coated with 0.1 mg/ml Poly-D-Lysine (Merck). After incubation for 60 min at 37 °C, non-adherent cells were removed and the medium was exchanged with MCM, supplemented with 10 ng/ml recombinant M-CSF and 2 ng/ml TGF-β (BioLegend). The medium was renewed every other day, and cells were stimulated at day 6 of culture. Stimulation included 300 U/ml IFN-γ, 500 U/ml TNF-α, 40 ng/ml IL-4 (all Peprotech), 10 ng/ml LPS (Sigma), or 10 µg/ml myelin debris (for preparation, see below).
