CD4 T Cell Recall Response to MOG

For CD4 T cell recall response, mice were immunized with 150 μg MOG_35–55 (American peptide) in 4 mg/ml CFA (Diffco) as described earlier (10 (link), 22 (link)). On day 7 post-immunization, CD4 T cells were isolated from draining lymph nodes and spleen using negative selection and magnetic beads and incubated in the presence of irradiated splenocytes, MOG_35–55 peptide (5–10 μg/ml) or stimulating anti-CD3 (BD Biosciences, clone 145-2C11) and anti-CD28 (BD Biosciences, clone 37.51) mAbs as described earlier (12 (link)). The experiments were performed in the presence of 30 μM of Forskolin or 0.1% DMSO in media (Control). After 72 h of incubation, the dead cells were removed using Ficoll™ gradient centrifugation, washed and stained for cells surface markers CD4 and TCRVβ₁₁, and were analyzed for BrdU incorporation by three-color flow cytometry.