Cx3cl1

Primary astrocytes and microglia were prepared from neonatal (P0-P2) mouse cortex from Cx3cr12^+/+ and Cx3cr1^-/- and grown and isolated as described in [20] . Briefly, neonatal (P0-P2) mouse cortex were mechanically dissociated and the cells were seeded onto 75 cm² flasks in DMEM:F12 supplemented with 10% FCS and penicillin/streptomycin. After 2 weeks in culture, flasks were trypsinized and separated using CD11b MicroBeads for magnetic cell sorting (MACS Miltenyi Biotec, Germany). Microglial and astroglial cultures were at least 99% pure, as judged by immunocytochemical criteria. Medium was changed to Dulbecco's Modified Eagle Medium:F12 (DMEM:F12) serum-free without antibiotics 16 h before treatment. Immortalized microglial cell line (IMG) isolated from the brains of adult mice, were purchased from Kerafast Inc., and were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and 2 mM L-glutamine, in 5% CO₂ at 37 °C, 50% relative humidity. Medium was changed to serum-free DMEM without antibiotics 16 h before treatments. CX3CL1 was obtained from PreproTech (Catalog# 400-26) and solubilized in water at 46 μM and used at 100 nM. Sulforaphane was obtained from LKT Labs (Catalog# S8044) and used at 15 μM for short-time treatment (6 h) or 5 μM for long-time treatment (16 h).

Human iPSC-derived microglia cells were generated from the BIONi010-C human iPSC line (EBiSC), originating from a non-demented, normal male (15–19 years old). Differentiation to human iPSC-derived microglia was essentially as described by Xiang et al.⁷⁸ (link) Briefly, the protocol was as follows: embryoid body differentiation media for 2 days (consisted of Essential 8^TM, 50 ng/ml BMP4, 50 ng/ml VEGF, 20 ng/ml SCF, and 10 µM Y-27632), then myeloid differentiation media for 30 days [consisted of X-VIVO^TM 15 medium (Lonza), GlutaMAX^TM (Life Technologies), 100 U/ml penicillin/streptomycin (Life Technologies), 50 µM β-mercaptoethanol (Life Technologies), 100 ng/ml MCSF (Peprotech), and 25 ng/ml IL-3 (Cell Guidance Systems)]. These myeloid cells were seeded at a density of 5 × 10⁵ cells/well in six-well plates, followed by microglial differentiation media for 13 days [consisted of: DMEM/F12 HEPES no phenol red, 2% ITS-G (Life Technologies), 1% N2 supplement (Life Technologies), 200 µM monothioglycerol (Sigma), GlutaMAX^TM, NEAA (Life Technologies), 5 µg/ml insulin (Sigma), 100 ng/ml IL-34 (Peprotech), 25 ng/ml MCSF and 5 ng/ml TGFβ-1 (Peprotech)] and finally, microglia maturation media for 4 days [consisted of: microglial differentiation media plus 100 ng/ml CD200 (Generon), and 100 ng/ml CX3CL1 (Peprotech)].