Chondrocyte-Macrophage Co-culture Proliferation Assay

Chondrocytes were seeded into 24-well plates and cultured to achieve 70–80% confluence for use in proliferation assay. THP-1 cells (1.6 × 10⁴ cells/cm²) were seeded in the upper layer of a 24-well transwell chamber (pore size: 0.4 μm, Corning) whereas PBS buffer was added to the lower chamber. Adherence of THP-1 was induced by PMA. THP-1 cells were then transfected with siRNA-NC or siRNA-VDR. After transfection, cells were treated with complete culture medium containing LPS for 4 h and then treated with complete culture medium containing LPS & Nig for 1 h. Cell proliferation was analyzed using EdU kit.41 (link) We transferred the transwell chamber into the chondrocytes-adhered 24-well plates to establish the co-culture system and the medium containing the LPS, Nig and EdU (10 μM) was replaced; cells were then incubated for 24 h. After incubation, BeyoClick™EdU-594 Cell Proliferation Detection Kit (Beyotime) was used to determine proliferation rate following the manufacturer’s instructions. Cell proliferation of each group was analyzed by fluorescence microscope and flow cytometry.