Sytox blue live dead stain

Brain tissues were collected from perfused male Mg^WT and Mg^PTX mice as previously described^{41 (link)}. Single-cell suspensions were prepared from tissue containing cortical and hippocampal regions following the adult brain dissociation (ABD) kit manufacturer’s instructions with modification (Miltenyi Biotec). Briefly, minced tissues were incubated with ABD Mix 1 for 15 min at 34 °C, and then ABD Mix 2 was added for 10 min at 34 °C. Tissues were gently triturated and then incubated for 10 min at 34°C. Homogenized tissue solutions were passed through 70-μm smartstrainer (Miltenyi Biotec), washed with cold Dulbecco’s PBS and centrifuged at 450xg for 7 min at 4°C. Tissue debris was removed following the ABD Kit debris removal step, followed by straining through 30-μm smartstrainer (Miltenyi Biotec) and then centrifuged at 450xg for 7 min at 4°C. Single-cell suspensions were incubated with 1 μM Sytox blue live/dead stain (Thermo Fisher Scientific) for 5 min at 4°C and then cell sorting was performed on an FACSARIA II (BD Biosciences). Live Sytox blue⁻ CX3CR1-GFP⁺ cells were sorted directly into tubes containing RLT plus lysis buffer (Qiagen) supplemented with 1% 2-mercaptoenthanol (Sigma) and 0.25% reagent DX (Qiagen). Cell lysates were frozen on dry ice and immediately stored at −80°C until use.