Rhil 6

For calcification staining, Alizarin red S method was used. Cells cultured with rhIL-6 50 ug/L (R&D Systems) for six days served as the experimental group and the routinely cultured cells as the control group. Cells were washed with D-Hank’s solution, fixed with pure iced ethanol, and were then exposed to 0.5% Alizarin red S (PH 7.0) for 15 min at room temperature (red/orange means a positive staining of extracellular matrix). For calcium concentration measuring, O-cresolphthalein complexone method was used. Cells cultured with rhIL-6 10 ug/L or 50 ug/L served as experimental groups and routinely cultured cells served as controls. The calcium measuring was applied at baseline and after being treated for 3, 6, 9, and 12 days. Total calcium in the cell layer was extracted by 0.6 mol/L HCl solution for 24 h at 37°C. The remaining components of cells were dissociated by 1 mL of 0.1 N NaOH/0.1% SDS to obtain the total protein. Protein concentrations were determined using a BCA protein assay kit (Pierce, Cat. no.23227) and final concentrations of calcium were adjusted by the protein content of the cell layer.

Purified naive and total human CD8⁺ T cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% L-glutamine, respectively. Cells were seeded at 5 to 10×10⁴ cells into 96-well U bottom plate and stimulated with anti-CD3/CD28 coated microbeads (Dynabeads® T-Activator CD3/CD28; Thermo Fisher Scientific, Waltham, MA, USA) in the absence or presence of the indicated cytokines; recombinant human (rh) IL-1β (25 ng/ml), rhIL-6 (6.25 to 100 ng/ml), rhIL-21 (0.1 to 100 ng/ml), rhIL-7 (50 ng/ml), IFN-α (50 or 100 ng/ml), rhIL-10 (6.25 to 100 ng/ml; all from R&D systems, Minneapolis, MN, USA), rhIL-12 (50 ng/ml), rhIL-15 (50 or 100 ng/ml), rhIL-2 (100 IU/ml, all from PeproTech, Rocky Hill, NJ, USA). In some experiments, cells were stimulated with anti-CD3/CD28 coated microbeads with chemical inhibitors: STAT1 inhibitor Fludarabin, STAT3 inhibitor 5,15-DPP, or STAT5 inhibitor CAS 285986-31-4 (all from Sigma-aldrich, St. Louis, MO, USA).