Human OA chondrocytes, at the first passage, were plated in 6-well dishes at a starting density of 1 × 105 cells/well until they became confluent. Human recombinant visfatin (Sigma–Aldrich, Milan, Italy) was first dissolved in phosphate buffered saline (PBS) (Euroclone, Milan, Italy), according to the manufacturer’s instructions and then it was diluted in the culture medium immediately before the treatment to reach the final concentration required.
The cells were cultured for 24 h in DMEM with 0.5% FBS and visfatin at concentration of 5 μg/mL and 10 μg/mL. The concentrations of the adipokine used in this in vitro study were selected according to those used by other authors [13 (link),18 (link)]; the final concentrations were chosen based on the best results obtained in terms of viability.
Afterwards, 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.
After the treatment, the media were removed, centrifuged and stored at −80 °C, while the cells were immediately processed to carry out flow cytometry analysis and quantitative real-time PCR.
The cells were cultured for 24 h in DMEM with 0.5% FBS and visfatin at concentration of 5 μg/mL and 10 μg/mL. The concentrations of the adipokine used in this in vitro study were selected according to those used by other authors [13 (link),18 (link)]; the final concentrations were chosen based on the best results obtained in terms of viability.
Afterwards, 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.
After the treatment, the media were removed, centrifuged and stored at −80 °C, while the cells were immediately processed to carry out flow cytometry analysis and quantitative real-time PCR.
Free full text: Click here
