Brain Tissue Cell Isolation and Characterization

Cells were isolated from brain tissue as previously described [20 (link)], with some modifications. Briefly, isolated tissues were cut up into fine pieces and were dissociated in buffer containing HBSS (without calcium/magnesium), 5% FBS, 10 μM HEPES, 2 mg/mL collagenase D (Sigma-Aldrich) and 28U/mL DNaseI (NEB) at 37 °C for 45 min. Every 15 min, dissociated tissue was pipetted up and down using sequentially smaller Pasteur pipettes to homogenize tissue debris. Homogenate was filtered through a 70-μm filter and centrifuged 10 min at 300 g. Pellets were resuspended in PBS and stained with an APC-Cy7 LIVE/DEAD™ fixable dead cell stain kit (Invitrogen). Cells were washed, resuspended in FACS buffer with Fc block (BD Biosciences) and stained with the following antibodies: AF700 anti-CD3 (BioLegend, clone:17A2), BV711 anti-Ly6C (BioLegend, clone: 1A8), BV421 anti-Ly6G (BioLegend, clone: HK1.4), FITC anti-CX3CR1 (BioLegend, clone: SA011F11), BV510 anti-CD19 (BioLegend, clone: 6D5), PerCPCy5.5 anti-CD45 (BioLegend, clone: 30-F11), and APC anti-Cd11b (BioLegend, clone: M1/70). Samples were measured on an LSRFortessa (BD Biosciences) and analyzed using FlowJo software.