We followed the methods reported by Xu et al. for cell culture [21 (link)]. A 6-well assay plate precoated with anti-CD3 and anti-CD28 Ab (BD Pharmingen) was incubated at 37 °C for 4 h. Splenocytes (1 × 106 cells/well) were obtained from the spleens of 3 to 4-weeks-old mice and cultured for 3 d in 6-well plates in a medium containing TGF-β (2 ng/mL; Peprotech), IL-6 (20 ng/mL; Peprotech), anti-IFN-γ (10 μg/mL; eBioscience), and anti-IL-4 (10 μg/mL; eBioscience). These cells were treated with DMSO or Am80 (1 or 10 nmol/L) for 72 h and then collected for the Th17 cell differentiation test using flow cytometry, while the cell supernatants were used for the detection of IL-17A using enzyme-linked immunosorbent assay (ELISA).
The rat renal tubular epithelial cell line, NRK-52E, was purchased from GuangZhou Jennio Biotech and cultured in F12/DMEM (Hyclone) with 10% fetal calf serum (GIBCO). After pre-incubation in F12/DMEM without serum for 8 h, the NRK-52E cells were treated with PBS, TGF-β (10 ng/mL; Peprotech), IL-17A (10 ng/mL; Peprotech), or TGF-β (10 ng/mL) + IL-17A (10 ng/mL) for 48 h.
The rat renal tubular epithelial cell line, NRK-52E, was purchased from GuangZhou Jennio Biotech and cultured in F12/DMEM (Hyclone) with 10% fetal calf serum (GIBCO). After pre-incubation in F12/DMEM without serum for 8 h, the NRK-52E cells were treated with PBS, TGF-β (10 ng/mL; Peprotech), IL-17A (10 ng/mL; Peprotech), or TGF-β (10 ng/mL) + IL-17A (10 ng/mL) for 48 h.