Cd11b microbead isolation kit

Mice aged P0–P3 were euthanized by decapitation, and brains were dissected into PBS on ice. Brains of 6–8 mice were pooled, centrifuged at 500 × g for 10 min at 4 °C and resuspended in 0.25% Trypsin-EDTA (Thermo Fisher Scientific) at 37 °C for 10 min. DNase I (Thermo Fisher Scientific) was added at 1 mg/ml to the solution, and the brains were digested for 10 more minutes at 37 °C. Trypsin was neutralized by adding DMEM + GlutaMAX (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific) and 1% penicillin/streptomycin (Thermo Fisher Scientific), and cells were passed through a 70 µm cell strainer. Cells were centrifuged at 500 × g for 10 min at 4 C, resuspended in DMEM + GlutaMAX with 10% FBS 1% penicillin/streptomycin and cultured in T-75 flasks (Sarstedt), pre-coated with 2 µg/ml Poly-L Lysine (PLL, Provitro) at 37 °C in a humidified incubator with 5% CO₂ for 5–7 days until confluence was reached. Mixed glial cells were shaken for 30 min at 180 rpm, the supernatant was collected and the medium was changed, and then cells were shaken for at least 2 h at 220 rpm and the supernatant was collected and the medium was changed again. CD11b+ microglia were isolated from the collected supernatant using the CD11b Microbead Isolation kit (Miltenyi) according to the manufacturer’s instruction and seeded onto PLL-coated plates.

Spleens, femurs, tibias, and fibulas were taken from C57BL/6 mice to obtain CD11b⁺ cells. Single-cell suspensions were enriched using a CD11b MicroBead isolation kit (Miltenyi Biotec) to obtain myeloid cells. For positive selection, the autoMACS Pro Separator (Miltenyi) was used. Pooled CD11b⁺ cells from the bone marrow and spleens (purity > 97%) were resuspended in PBS, and 4 × 10⁶ cells were injected into the retroorbital sinus of the recipient C57BL/6 mouse.