Macs column system

Spleens were mashed and both blood and spleen cells were separated based on Ter119 using a biotinylated anti-Ter119 antibody (Key resources table) and anti-biotinylated beads on the MACS column system (Miltenyi). Ter119⁺ cells were stained for Ter119 and CD44 (Chen et al., 2009 (link)). Ter119^- cells were stained for CD45.1 CD11b, CD11c, CD19, and, if appropriate, CD115, Siglec-F, and Ly6G (Key resources table). Bone marrow cells were flushed from bones and enriched for C-Kit⁺ cells as above. When appropriate, the C-Kit^- fraction was further separated based on Ter119 and stained as above. C-Kit⁺ cells were stained for C-Kit, Flt3, CD150, Sca-1, and, if appropriate, CD41 (Key resources table), and PI (1:5000) as described above. For analyzed and/or sorted populations, see Figure 1—figure supplement 1. Populations were only sorted for mice with an engraftment (donor cells percentage) of above 5% in spleen, bone, and blood.

5‐ and 18‐month‐old C57BL/6 male and female mice (4–5 animals per experimental group) were anesthetized with pentobarbital (Dolethal, 50 mg/kg body weight, intraperitoneal) and perfused transcardially with 0.9% saline. Adult or aged microglia were isolated as previously described (Lee & Tansey, 2013). Brain tissue was minced and digested at 37°C for 30 min with gentle shaking in a buffer containing papain, dispase II, and DNase I followed by mechanical dissociation. After neutralization of the reaction, cells were centrifuged and filtered by a 40‐µm mesh. Microglia fraction was obtained in a 30%–70% SIP Percoll gradient by centrifugation of the cells at 500g and 18°C for 30 min, with no brake. After centrifugation, myelin was discarded and 6ml of the interphase was collected and mixed with 40 ml RPMI. The cells were centrifuged at 500g and 18°C for 7 min with regular brake, and the pellet was resuspended on 2ml RPMI. After counting the cells, they were centrifuged at 168g for 10 min and resuspended in 180μL MACS buffer. Afterward, 10 μl/10⁷ cells of CD11b microbeads were added to the mix. After washing, magnetically labeled cells were collected using MACS column system (Miltenyi Biotec).

CD4⁺ T-cells from male Lewis rats were obtained from splenocytes and used in Th17 differentiation assays as previously described [23 (link)]. In brief, cells were incubated with the following rat-specific antibodies: CD8a (clone OX8; BioLegend), CD45RA (clone OX33; BioLegend), CD11b/c (clone OX42; BD Biosciences), CD25 (clone OX39; eBioscience), and erythrocytes (clone OX83; Cedarlane), followed by a washing step. Cells were incubated with anti-IgG coated microbeads (Miltenyi Biotec) and unwanted cells were magnetically depleted using a MACS column system according to the manufacturer`s instructions (Miltenyi Biotec). CD4<sup>+</sup> T-cells were stimulated with rat-specific anti-CD3 (pre-coated to the plated at 4 μg/ml, clone G4.18) and soluble anti-CD28 antibody (2 μg/ml, clone JJ319, BioXcell) together with recombinant human TGF-β1 (5 ng/ml, BioLegend), mouse IL-6 (20 ng/ml, BD Pharmingen), rat IL-1β (10 ng/ml, R&D Systems), and mouse IL-23 (10 ng/ml, BioLegend) in the presence of anti-rat IFN-γ antibody (5 μg/ml, BioLegend). After 72 hrs of incubation, the supernatants were harvested and IL-17A cytokine concentrations were quantified by ELISA according to the manufacturer`s specifications (eBioscience).