2.3. Culture of Primary Mouse Microglia

Primary microglia were isolated from the brains of newborn C57BL/6J mice (<seven days of age) using magnetic cell sorting with anti-CD11b antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany) as previously described [28]. CD11b-positive cells (> 90% purely evaluated using flow cytometry) were plated in poly-D-lysine (PDL)-coated 96-well plates (BD Biosciences, Billerica, MA, USA) and cultured in DMEM/F-12 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal calf serum (Gibco) and 100 U/mL penicillin/streptomycin (Sigma-Aldrich). Microglia isolated from newborn C57BL/6J mice were plated at 3 × 10⁴ cells/well in a PDL-coated plate.

To measure the production of inflammatory cytokines released into the culture medium, microglia were treated with each peptide for 12 h and then with lipopolysaccharide (LPS) (5 ng/mL; Sigma-Aldrich, Saint Louis, MO, USA) and interferon-γ (IFN-γ) (0.5 ng/mL, R&D Systems, Minneapolis, MN, USA) for 12 h. After treatment, supernatants were tested using an enzyme-linked immunosorbent assay (ELISA) (eBioscience, San Diego, CA, USA) or a multiplex cytokine assay (Bio-Rad, Hercules, CA, USA).

To measure the expression of cell surface markers, microglia were treated with a peptide and then with 5 ng/mL LPS and 0.5 ng/mL IFN-γ. The cells were incubated with the antibodies as follows: Anti-CD86-PE (clone; B7-2, eBioscience), anti-CD206-FITC (clone; C068C2, BioLegend, San Diego, CA, USA), and anti-CD11b-APC-Cy7 (clone; M1/70, BD Biosciences). Flow cytometry was performed using a FACSCanto II flow cytometer (BD Biosciences). Each sample was assayed in triplicate.