Anti cd80 clone 16 10a1

Microglia cells were isolated through Percoll gradient or Miltenyi Adult Brain Dissociation kit, as described in RNA‐Seq Analysis. Microglia staining occurred in 1X dPBS^−/− (Thermo Fischer) supplemented with 0.5% FBS, 0.4% 0.5 M EDTA, and 1% HEPES at 4°C for 20 min. Cells were stained with the following antibodies: anti‐CD11b (clone:M1/70, eBioscience), anti‐CD45 (clone:30‐F11, eBioscience), anti‐F480 (clone:BM8, eBiosience), anti‐CX3CR1 (clone:SA011F11, Biolegend), anti‐CD31 (clone:MEC13.3, Biolegend), anti‐CCR3 (clone:J073E5, Biolegend), anti‐ I‐A/I‐E (clone:M5/114.15.2, Biolegend), anti‐CD80 (clone:16‐10A1, eBioscience), anti‐CD86 (clone:GL1, eBioscience), and anti‐TLR4 (clone:SA15‐21, Biolegend). Following staining, cells were washed twice and fixed in a 1:1 solution of supplemented dPBS^−/−: 4% paraformaldehyde overnight at 4°C. After fixation, cells were resuspended in supplemented dPBS^−/−and enumerated via flow cytometry (BD LSR II with 561 laser). Microglia populations were identified as CD11b^high/CD45^low. Subsequent data was analyzed using FlowJo software (v. 10.5.3).

Flow cytometry was used to measure the phenotypical changes in Raw264.7 macrophages. Single-cell suspension is prepared before staining with fluorochrome-labeled anti-CDF4/80 (clone BM8, BioLegend, San Diego, CA, USA), anti-CD80 (clone 16-10A1, eBioscience, San Diego, CA, USA). Data were analyzed using FlowJo v.10 (Treestar, Ashland, OR).