Isolation of Lymphocytes and Neural Cells

Cell extraction from tissues was modified according to the described protocol60 (link). For lymphocyte extraction, organs were digested for 30–60 minutes at 37 °C, and shaken at 240 rpm in HBSS containing 1 mg/ml Collagenase Type I (Life Technologies). Digested slurry was filtered and washed through a 70 μM mesh filter and resuspended in Percoll (GE Healthcare) diluted to 90% using HBSS. Layers of 60% and 37% Percoll were then sequentially overlayed on the 90% cell-Percoll mix. Cell separation was accomplished by centrifugation at 4 °C, 500 × g for 18 minutes with the brakes off. Cells were washed and resuspended in staining buffer containing 5% FBS in PBS in preparation for flow cytometry. Neural cells extraction was performed using previously described methods60 (link). Briefly, cells were digested using 0.8 mg/ml Dispase 2 (Sigma), 0.2 mg/ml collagenase P (Sigma), and 0.1 mg/ml DNase I (Sigma). Density separation of cells was done in one step using only 40% Percoll, and subsequent pelleted cells were filtered and washed through a 100 μM filter before preparation for flow cytometry.