MLR Assay

CD4⁺ and CD8⁺ T cells were enriched by immunomagnetic negative selection from spleens of Balb/c mice using beads labeled with CD11b, CD11c, CD19, CD45R, CD49b, CD105, MHC-II, Ter-119, and TCR/γδ (Miltenyi Biotec) and MACS separation columns (Miltenyi Biotec). Isolated CD4⁺ and CD8⁺ T cells were labeled with 10 µM carboxyfluorescein diacetate succinimidyl ester (CFSE) (Thermo Fisher Scientific, Waltham, MA, USA). BMDCs (5 × 10³) were treated with LPS and inotodiol in a 96-well plate (Costar, Cambridge, MA, USA) for 24 h, after which CFSE-labeled T cells (1 × 10⁵) were added at a DC/T cell ratio of 1:20 and the cells were cultured at 37^°C in the presence of 5% CO₂ for 4 days. Cell-free supernatants were obtained and examined for cytokine secretion. T cell proliferation was measured based on the percentage of CFSE dilution.