Model 450 microplate reader

The viability of the chondrocyte culture was assessed using a CCK8 assay (Dojindo Molecular Technologies, Japan). The chondrocytes were re-suspended with a density of 2×10³ cells/mL in 96-well plates, and treated with CGA at different concentrations (0, 15, 30, 60, 120, and 240 μM). After 24, 48, and 72 hours, 10 μL of CCK8 (5 g/L) was added, followed by 3 hours of incubation at 37°C. The absorbance values were then measured at 450 nm using a Bio-Rad Model 450 Microplate Reader (Bio-Rad, USA). Cell viability was indirectly established using the ratio of the absorbance value of CGA-treated cells relative to the control. The final results were representative of three independent experiments.

Dihydroethidium (DHE) staining was performed at the end of cell culture using DHE fluorescent probe (Beyotime, Shanghai, China) to detect the presence of superoxide anion (O₂⁻). The cells were incubated with 10 μM DHE for 30 min, at 37 °C, and then collected for analyses according to the manufacturer’s instructions. HUVECs (control, 6 μM Baicalin, 50 mM glucose and 6 μM Baicalin+50 mM glucose group) were seeded into 96-well plates. These cells (1 × 10⁶ cells/ml) were maintained in DMEM+10% fetal bovine serum at 37 °C and 5% CO₂. The cell viability was assessed using CCK8 assay (cholecystokinin-8). Briefly, 10 μl of CCK8 reagent (Dojindo, Kumamoto, Japan) was added to the 96-well plates and incubated continually for 6, 12, 24 and 48 h at 37 °C. The absorbance values were measured at 450 nm using a Bio-Rad model 450 microplate reader (Bio-Rad, Hercules, CA, USA). The cell viability was indirectly determined by examining the ratio of the absorbance value of Baicalin or/and glucose-treated cells relative to the control cells, from this experiments.

Cell viability was determined with the WST-1 Cell Proliferation Kit (Takara), according to the manufacturer’s instructions. Briefly, 0.5–2 × 10⁴ cells in 96-well plates were infected with rMV-EGFP-SLAMblind and cultured for the indicated days in FBS-containing medium. For the evaluation of cell viability, 10 μl WST-1 solution was added in each well, incubated for 2–4 h, and then the absorbance at 450 nm was measured at the indicated dpi using a Model 450 Microplate Reader (Bio-Rad, Hercules, CA). The viability of the cells was determined as described previously13 (link).

Serum antibodies to OVA, type II collagen, Hsp65, and mBSA were assessed by ELISA. Briefly, 96-well microtiter plates (NUNC) were coated with a solution of OVA (5 μg/μL), CII (5 μg/μL), mBSA (2 μg/μL), or M. tuberculosis Hsp65 (1 μg/μL) overnight at 4°C. Plates were incubated with serum samples and bound antibodies detected using alkaline phosphatase conjugated goat anti-mouse IgG (Southern Biotechnology). Color reaction was developed at room temperature with orthophenylenediamine (OPD; 1 mg/mL), 0.04% H₂O₂ substrate in sodium citrate buffer. Reaction was interrupted by the addition of 20 μL/well of 2N H₂SO₄. Absorbance was measured at 492 nm by an ELISA reader (Bio-Rad Model 450 Microplate Reader). Results were calculated using the running sum of ODs of 6 dilutions starting at 1:100 and ending at 1:102,400 (6 serial 1:4 dilutions). This methodology represents more precisely antibody titers as previously described by our group (38 (link)). Alternatively, concentration of IgG1 anti OVA were obtained by interpolating a standard curve obtained with anti Ova IgG1 antibody (monoclonal OVA-14, Sigma). Rheumatoid Factor were measured using a specific kit (MyBioSource).