Mitomycin c

Splenic mononuclear cells were isolated from BALB/c mice, and CD4⁺ T cells were purified by negative selection using magnetic beads and the specific kit, as described by the manufacturer’s instruction (Miltenyi Biotech, Auburn, CA, USA). BMDCs were stimulated with LPS alone or together with ALX at 2 or 10 μM for 48 h; these cells were purified by positive selection using magnetic beads (Miltenyi Biotech) and treated with mitomycin C (30 mg/L, Sangon Biotech, Shanghai, China) for 30 min before harvesting, and washed with PBS twice to terminate the influence of drug on T cell proliferation. The different groups of BMDCs were co-cultured in triplicate with 1 × 10⁵ CD4⁺ T cells at a ratio (DC:T) of 1:5, 1:10, or 1:20, respectively in 96-well plates for 3 days. The CD4⁺ T cells alone served as the control. The proliferation in individual groups of T cells was determined by CCK-8 assay. The proliferation index (PI) was calculated as PI = (co-culture well OD value-medium OD value)/(T cell well OD value-medium OD value) [40 (link)].

After 7 day culture, BMDCs were loaded with MOG (10 μg/ml) [37 (link)] for 24 h and stimulated with LPS (1 μg/ml) in the presence or absence of ALX (2 or 10 μM) for 48 h. In addition, some of BMDCs were pretreated with mitomycin C (30 μg/ml, Sangon Biotech) for 30 min. Splenic CD4+ T cells were isolated from the MOG35-55-immunized C57BL/6 mice by negative selection. The purified CD4+ T cells were co-cultured with different groups of DCs at a ratio of 5:1, respectively. The cells were cultured for 4 days, and the suspended T cells were stained with fluorescent-labeled anti-CD25 and anti-CD69 (eBioscience, San Diego, CA, USA) [38 , 39 (link)] to measure T cell activation; the cultured supernatants were collected for measuring the levels of IL-2 and IFNγ using specific kits (USCN LIFE). The T cell proliferation was determined by CCK-8 assay (US Everbright).