Anti cd3 anti cd28 abs

CD4⁺CD25⁻ T cells from C57BL/6 mice were sorted out using FACSAria III cell sorter. Then, cells (4 × 10⁵ cells/well) were cultured in 96-well plates (200 μl/well) in complete RPMI-1640 medium (Gibico, USA) containing 10% fetal bovine serum (Gibico), 100 U/ml penicillin and 100 μg/ml streptomycin, and stimulated with anti-CD3/anti-CD28 Abs (2.5 μg/ml) and IL-2 (10 ng/ml, Peprotech) in the absence or presence of TGF-β1 (positive control, 5 ng/ml, Peprotech) or shikonin (0.25 or 0.5 μM) for 4 days. The frequency of CD4⁺Foxp3⁺ Tregs was finally determined using a FACSCalibur.

FACS-sorted CD3⁺ T cells derived from C57BL/6 mice were labeled with 3 μM CFSE (Invitrogen, Germany) at room temperature without light for 15 min. Then, cells (4 × 10⁵ cells/well) were cultured in 96-well plates in complete RPMI-1640 medium as described above and stimulated with anti-CD3/anti-CD28 Abs (2.5 μg/ml) plus IL-2 (10 ng/ml, Peprotech) at 37°C with 5% CO₂ for 4 days. These cells were also treated with either shikonin (0.25 or 0.5 μM) or CsA (0.1 μM) for 4 days. Finally, cell proliferation was measured using a FACSCalibur. On the other hand, the levels of IFN-γ, IL-10, TGF-β1, and IL-17A in the supernatant were also measured using ELISA according to the manufacturer's instructions (Boster, China), and the absorbance was read at 450 nm in a microplate spectrophotometer (Thermo Fisher Scientific, USA).

T cell cytotoxicity was detected using CCK-8 assays. FACS-sorted CD3⁺ T cells derived from C57BL/6 mice were cultured in 96-well plates in complete RPMI-1640 medium (Gibico, USA) containing 10% fetal bovine serum (Gibico), 100 U/ml penicillin and 100 μg/ml streptomycin, and stimulated with anti-CD3/anti-CD28 Abs (2.5 μg/ml) and IL-2 (10 ng/ml, Peprotech). Shikonin was added to each well at different concentrations (0.1, 0.25, 0.5, 1, and 2 μM, respectively) with four wells per concentration. After 24, 48, and 96 h, 20 μL of CCK-8 was added to each well and incubated at 37°C for 4 h. The absorbance was measured by a microplate spectrophotometer (Thermo Fisher Scientific, USA) at the wavelength of 450 nm. Control without shikonin was set as 1.0.