Cfse labeling kits

Enriched T cells were stained with carboxyfluorescein succinimidyl ester (CFSE) using CFSE labeling kits (Molecular Probes, Eugene, OR, USA) according to the manufacturer’s instructions. CFSE-labeled cells were co-cultured with DCs as previously described. After 72 h, CFSE-labeled cells were detected in an FITC channel.

Splenocytes were isolated from wild type C57BL/6 mice spleen and disaggregated in RPMI 1640 medium. CD3⁺ T cells were isolated by passing the splenocytes through a nylon wool (Polysciences Inc., Warrington, PA, USA) column. Nylon-wool column-purified T cells from spleens were labeled using CFSE labeling kits (Molecular Probes, Oregon, USA) according to the manufacturer’s instructions. Cells were then co-cultured with DCs at a DC:T cell ratio of 1:10. CFSE-labeled purified CD3⁺ T cells (1 × 10⁶ cells/ml) were used as responders and imDCs or mDCs (1 × 10⁶ cells/ml) were used as stimulators. Cells were co-cultured at 37 °C for 72 h in 2 ml of RPMI 1640 supplemented with 10% FBS.