Magnetic Isolation and FACS sorting of WT and TREM2 KO ex vivo microglia

Dissociated cell pellets were resuspended in 160 μL FACS buffer (0.5% BSA in 1X DPBS) + 40 μL Mouse cell removal beads (Miltenyi) and incubated at 4 °C for 15 min. Magnetically labeled cells were then separated using LS columns and the MidiMACs separator (Miltenyi) while the unlabeled human cells were collected in the flow through. Isolated human cells were pelleted via centrifugation and prepared for cell sorting by resuspending in 400 μL of FACS buffer containing Hoescht 33342 (1:400) as a viability marker. For separation of human microglia expressing either endogenous GFP or RFP, samples were sorted on a FACS ARIA Fusion II (BD Biosciences) using the 70 um nozzle at the lowest flow rate. After removing doublets, by both forward and side scatter parameters, and selecting for live cells (Hoescht 33342^-), both GFP (GFP⁺RFP^-) and RFP (GFP^-RFP⁺) cells were gated on and sorted into individual tubes. For each cell type, 15,000 cells were sorted into collection tubes containing 5uL of FACS buffer, resulting in a final volume of ~20 μL.