Legendplex mu th cytokine panel

Serum cytokines were measured with the LEGENDplex™ MU Th Cytokine Panel (Biolegend) following the manufacturer’s instructions.

In the MLR, the simulator cells were saline-treated or Mn²⁺-treated murine BMDCs from BALB/c mice. The responder cells were splenocytes from C57BL/6 mice. Before the MLR, the stimulator cells were treated by Mitomycin-C (40 μg/ml, M5353, Sigma-Aldrich) for 30 min, and the responder cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (5 μM, 65–0850-84, ThermoFisher). The ratio of responder to stimulator was 2:1. Then, TGF-β1 and treatment Abs were added to the mixed cells. The mixed cells were cultured with RPMI-1640 containing 10% fetal bovine serum for 96 h. Subsequently, the mixed cells and supernatants were harvested to measure cell proliferation and cytokine levels. The ratios of daughter CD4⁺ and CD8⁺ T cells were determined by CFSE dilution assays (α-CD4: 100,516, BioLegend; α-CD8α: 563,068, BD Biosciences). The cytokine levels were measured by LEGENDplex™ MU Th Cytokine Panel (741,044, BioLegend).