Facsarial 3 cytometer

Fc receptors on isolated microglia were blocked with anti-CD16/CD32 antibody (BD Bioscience, 1:100). Thereafter, samples were incubated with anti-CD11b-APC (Biolegend, 1:800) and anti-CD45-PE (BD Bioscience; 1:400). Compensation was made with single stainings and gating was determined by proper negative isotype stained controls. Dead cells were excluded by a viability staining, PI (propidium iodide) (BD Bioscience). Flow cytometry was performed in a FACSArial III cytometer (BD Biosciences) and FACS Diva software (BD Biosciences). Microglia were identified by CD11b + and CD45 + expression and ten thousand events were recorded. Phagocytic microglia were recognized with Methoxy-X04 (Supplementary Figs. S6 and S7). FlowJo 10.4.2 software (Flowjo, LLC) was used for data analysis.

Microglia isolated by CD11b microbeads (Miltenyi Biotec) were analyzed for microglial cell surface antigens by flow cytometry. Briefly cells were incubated with anti-CD16/CD32 antibody (BD Bioscience, 1:100) to block Fc receptors. Samples were then incubated with anti-CD11b-APC (Biolegend, 1:800) and anti-CD45-PE (BD Bioscience; 1:400) to confirm purity of the microglial population. Gating was determined by proper negative isotype stained controls and compensation was made with single stainings. A viability staining, 7-aminoactinomycin D (7AAD)(BD Bioscience) was used to exclude dead cells. Flow cytometry was perfomed in a FACSArial III cytometer (BD Biosciences) and FACS Diva software (BD Biosciences). Ten thousand events were recorded and microglia were identified by CD11b⁺ and CD45⁺ expression^{12 (link)}. Data analysis was made using FlowJo 10.3 software (Three Star, Inc). Confirmed the enrichment of microglial cells by flow cytometry using CD11b and CD45 microglial markers (Supp. Figure 1).