Macs t cell isolation kits

Splenic CD8⁺, CD4⁺CD25⁻ or CD4⁺CD25⁺ T cells from wild-type, OX40^−/−, OT-I- or OT-II-transgenic mice were isolated by using the MACS T-cell isolation kits (Miltenyi Biotec). T-cell proliferation was measured with CFSE dilution assay. CD8⁺ or CD4⁺CD25⁻ T cells were incubated for 5 minutes at 37 °C with 1 μM CFSE in PBS, and then washed extensively. For antigenic stimulation using TCR-transgenic OT-I or OT-II T cells and antigen-pulsed DCs, 1 × 10⁵ purified T cells and 1 × 10⁴ DCs pulsed with OT-I or OT-II peptides (1 μg/ml) were seeded in each well of a round-bottom 96-well microtiter plate in triplicate in 200 ml RPMI-1640 medium supplemented with 10% FBS. For stimulation using wild-type or OX40^−/− T-cells, 1 × 10⁵ purified T cells and 1 × 10⁴ unpulsed DCs were cocultured in the presence of anti-CD3 antibodies (1 μg/ml). For suppression assays, 1–2 × 10⁵ unlabeled CD4⁺CD25⁺ Treg were added in the cocultures. Isotype-matched control IgG and blocking mAbs were added at a concentration of 10 μg/ml. We measured proliferation by relative CFSE dilution after 60 hours of culture. For human T cell proliferation, h-MoDCs were mixed with CFSE-labeled alloreactive CD4⁺CD25⁻ Teff in Aim-V medium, in the presence or absence of autologous CD4⁺CD25⁺ Treg. Human T cell proliferation was assessed 6 days after coculture.