Splenocyte Isolation

Spleens were harvested from EAE and healthy control mice on peak-of-disease or day 25 for in vitro and in vivo studies, respectively. Spleens were placed in 5 mL of RPMI 1640 containing L-glutamine and 1% Penicillin-Streptomycin and placed on ice for transportation. The spleens were pressed through sterile wire mesh with the use of a rubber 1 mL syringe plunger and the cellular extract was collected and centrifuged at 1100xg for 5 minutes. In order to lyse red blood cells the cell pellet was resuspended in 5 mL of Gey’s lysis solution for 5 minutes on ice. Quenching of the lysis solution was performed by adding 10 mL of RPMI 1640 supplemented with L-glutamine, 1% Penicillin-Streptomycin, and 10% fetal bovine serum (FBS) (complete RPMI, cRPMI). Cells were centrifuged at 1100xg for 5 minutes and resuspended in cRPMI prior to counting in 0.04% trypan blue. Cultures with in vitro treatments or PLP rechallenge were kept at 37°C and 5% CO₂.