T/C-28a2 cell line and human OA chondrocytes, at the first passage, were plated in 6-well dishes at a starting density of 6 × 106 cells/well until they became confluent. Human recombinant visfatin (Sigma–Aldrich, Milan, Italy) and human recombinant resistin (BioVendor, Rome, Italy) were first dissolved in phosphate buffered saline (PBS) (Euroclone, Milan, Italy), according to the manufacturer’s instructions, and then they were diluted in the culture medium immediately before the treatment to reach the final concentration required.
The cells were treated for 24 h with visfatin at concentration of 5 μg/mL and 10 μg/mL or resistin 50 ng/mL and 100 ng/mL. The concentrations of the adipokines used in our in vitro study were selected according to those used by other authors [12 (link),39 (link)]; the final concentrations were chosen based on the best results obtained in terms of viability.
After the treatment, the media were removed, centrifuged, and stored at −80 °C; the cells were immediately processed to carry out cell viability assay, flow cytometry analysis, and quantitative real-time PCR.
Afterwards, the cells were pre-incubated for 2 h with 1 μM BAY 11-7082 (NF-κB inhibitor, IKKα/β, Sigma–Aldrich, Milan, Italy) and then treated 24 h with the tested concentrations of visfatin and resistin. Subsequently, the gene expression of MMP-1, MMP-13, and Col2a1 was evaluated.
The cells were treated for 24 h with visfatin at concentration of 5 μg/mL and 10 μg/mL or resistin 50 ng/mL and 100 ng/mL. The concentrations of the adipokines used in our in vitro study were selected according to those used by other authors [12 (link),39 (link)]; the final concentrations were chosen based on the best results obtained in terms of viability.
After the treatment, the media were removed, centrifuged, and stored at −80 °C; the cells were immediately processed to carry out cell viability assay, flow cytometry analysis, and quantitative real-time PCR.
Afterwards, the cells were pre-incubated for 2 h with 1 μM BAY 11-7082 (NF-κB inhibitor, IKKα/β, Sigma–Aldrich, Milan, Italy) and then treated 24 h with the tested concentrations of visfatin and resistin. Subsequently, the gene expression of MMP-1, MMP-13, and Col2a1 was evaluated.
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