Live dead amine reactive violet viability marker

Brains were digested in RPMI containing 1.6 mg/mL collagenase (type IV; Sigma-Aldrich) and 200 µg/mL DNase I (Sigma-Aldrich) at 37 °C for 50 min. Cells were isolated using a Percoll gradient (GE Healthcare) and debris was filtered out using a 70 µm nylon mesh. Cells were counted and labelled with LIVE/DEAD amine-reactive violet viability marker according to the manufacturer’s protocol (Invitrogen). Cells were labeled with FITC anti-CD45 (eBioscience; 30-F11), PE anti-CD11b (BD Pharmingen; M1/70), PerCP-Cy5.5 anti-CD4 (eBioscience; RM4–5) and APC-eFluor780 anti-CD8 (eBioscience; 53–6.7). Flow cytometry was performed using a BD LSR Fortessa and results were analyzed using FlowJo version 9.6.2. Mice were perfused for the analysis of brain sequestered cells.

Flow cytometry was performed using a BD LSR Fortessa and results were analyzed using FlowJo version 9.6.2. Mice were perfused for the analysis of brain sequestered cells. Brains were digested in RPMI containing 1.6mg/mL collagenase (type IV; Sigma-Aldrich) and 200μg/mL DNase I (Sigma-Aldrich) at 37°C for 50 min. Cells were isolated using a Percoll gradient (GE Healthcare) and debris was filtered out using a 70μm nylon mesh. Cells were counted and labelled with LIVE/DEAD amine-reactive violet viability marker according to the manufacturer’s protocol (Invitrogen). The cells were blocked and labeled with FITC anti-CD45 (eBioscience; 30-F11), PE anti-CD11b (BD Pharmingen; M1/70), APC anti-CD4 (eBioscience; RM4-5) and PerCP-Cy5.5 anti-CD8 (eBioscience; 53–6.7).