Betaplate1205

Draining lymph nodes of rats 10 days after immunization were dissected under sterile conditions and passed through a 40-μm cell strainer. The derived cell suspension was cultured in flat bottom 96-well plates in standard T cell medium (RPMI 1640 with 5% FCS, 2 mM l-glutamine and 50 μM 2-ME, life technologies, ThermoFisher). P2_53–78 was added to each well in concentration from 0 to 100 μg/ml. T cell proliferation was measured via [3H] thymidine incorporation during the last 24 h of a 3-day incubation. Results from liquid scintillation counting (BetaPlate1205, Perkin Elmer, Boston, USA) are given as mean counts per minute (cpm) of quadruplicate cultures ± SEM. A stimulation index was calculated as the ratio of cpm at indicated P2_53–78 concentrations to proliferation in the absence of antigen.

Spleens of mice were dissected under sterile conditions and passed through a 40 μm cell strainer followed by ammonium chloride based erythrocyte lysis (BD Biosciences, Germany). Derived splenocytes were cultured in flat bottom 96-well plates in standard T cell medium (IMDM with 5% FCS, 2 mM L-glutamine and 50 μM 2-ME, life technologies). To restimulate cells, MOG_35-55 was added during the culture period with increasing concentration from 0 to 100 μg/ml. T cell proliferation was measured via [³H] thymidine incorporation during the last 24 h of a four day incubation. Counts per minute (cpm) of quadruplicate test cultures ± SEM were determined using liquid scintillation counting (BetaPlate1205, Perkin Elmer, Boston, US). Stimulation index was calculated as ratio of the cpm at the indicated MOG_35-55 concentrations to the proliferation of cells in the absence of MOG_35-55. Flow cytometry was performed from homogenized lymphoid organs. Cells were stained for cell surface CD4 (L3T4, APC or Pacific Blue labelled), CD25 (3C7, PerCP labelled) and intracellular FoxP3 (MF23, Pacific Blue labelled) using the FoxP3 staining buffer set (all from BD Biosciences). Flow cytometry was performed using a FACSCanto II flow cytometer (BD Biosciences).