2.5.2. Intracellular Oxidative Stress

DCFH-DA assay was used to measure the production of intracellular reactive oxygen species (ROS) in C20A4 cells according to the manufacturer's protocol. Following treatment, cells were labeled with DCFH-DA (25 μM) for 1 h in the dark. The fluorescence was measured every 5 min for 1 h, with an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a microplate reader (Cytation 3).

The malondialdehyde (MDA) concentration, as a lipid peroxidation index, was determined using the thiobarbituric acid reactive substances (TBARS) assay, according to the manufacturer's protocol. The basal concentration of MDA was established adding about 600 μL of TBARS solution to 50 μg of total protein dissolved in 300 μL of Milli-Q water. The mix was incubated for 40 min at 100 ◦C prior to centrifugation at 14,000 rpm for 2 min. The supernatant was analyzed with a microplate reader at a wavelength of 532 nm [39].

Total SOD-like activity was assessed with the SOD Assay Kit-WST according to the manufacturer's protocol. The activity was expressed as units per mg of protein, where one unit of enzyme inhibits reduction of cytochrome C by 50% in a coupled system formed by xanthine and xanthine oxidase.

## 2.5.3. Enzyme-Linked Immunosorbent Assay (ELISA)

Secreted IL-6, IL-8, and TNF-α protein levels were measured in supernatants of chondrocytes treated as reported in paragraph 2.5.1. Briefly, 100 μL of samples and standards were added into the wells already pre-coated with antibody specific for IL-6, IL-8, or TNF-α, and incubated for 2 h at 37 ◦C. Unbound substances were removed and 100 μL of biotinconjugated antibody specific for IL-6, IL-8, or TNF-α was added to the well. After washing, 100 μL of avidin conjugated Horseradish Peroxidase (HRP) was added to the wells and incubated for 1 h at 37 ◦C, followed by addition of 90 μL of TMB substrate solution, and then incubation for 15–30 min at 37 ◦C. Stop solution was added to each well, the plate was gently tapped for thorough mixing, and the color intensity measured at 450 nm using a Cytation 3 Microplate Reader (ASHI, Milan, Italy).
