2.4g2 hybridoma

Brains and spinal cords were removed from mice after perfusion with Hank’s balanced salt solution (HBSS) through the left ventricle. The tissues were forced through steel mesh to prepare single-cell suspensions and incubated at 37° C for 45 min in 250 μg/ml collagenase type 4 (Worthington Biochemical Corp., Lakewood, NJ, USA). A continuous 100% Percoll gradient (Pharmacia, Piscataway, NJ, USA) was constructed at 27,000 x g for 30 min to enrich for CNS-infiltrating mononuclear cells. CNS mononuclear cells from mice were cultured in plates coated with either PMA plus ionomycine or viral epitope peptides in the presence of Golgi-Plug for 6 h at 37°C. The cells were then incubated in 50 μl of 2.4G2 hybridoma (American Type Culture Collection, Manassas, VA, USA) supernatant for 30 min at 4°C to block the Fc receptors. The cells were incubated for an additional 30 min at 4°C in the presence of allophycocyanin-conjugated anti-CD8 (clone 53–6.7) or anti-CD4 (GK1.5) antibodies diluted in 50 μl of Fc-block (2.4G2 supernatant). After two washes, intracellular IFN-γ staining was performed according to the manufacturer's instructions (BD Pharmingen, San Diego, CA, USA) using phycoerythrin-labeled rat monoclonal anti-IFN-γ antibody (XMG1.2). The cells were analyzed on a Becton Dickinson FACSCalibur flow cytometer.