Bms224 2

Samples of synovial fluids were obtained from patients with hip OA and femoral neck fracture during hip arthroplasty surgery. ELISA (enzyme-linked immunosorbent assay) was used to measure IL-1β (#BMS224-2, Invitrogen, Carlsbad, CA, USA) and TNF-α (#BMS223-4, Invitrogen, Carlsbad, CA, USA) levels in synovial fluids according to the manufacturer’s instructions.

Cells were lysed in RIPA buffer with 10% protease inhibitor (11873580001 cOmplete™, EDTA-free Protease Inhibitor Cocktail, Roche). Protein concentration was determined using the Bradford assay (B6916, Sigma). For Western blot analysis, 50 µg of lysate was separated by electrophoresis using PAGE gels (NuPAGE 4–12% Bis–Tris Gel, Thermo Fisher) and transferred to PVDF membranes (Amersham Hybond P 0.45 PVDF 10600023). After blocking with 5% non-fat dried milk, membranes were incubated overnight at 4 °C with the following primary antibodies: anti-IL-8 (ab110727; Abcam, dilution 1:1000), anti-IL-6 (#12153; Cell Signalling, 1:1000), and anti-β-actin (#8457S; Cell Signalling, 1:1000). Secondary IgG HP-conjugated anti-rabbit HRP-linked antibody (#7074; Cell Signalling, 1:3000) were applied for 1 h at room temperature. Immunoreactive proteins were revealed with SuperSignal™ West Pico (ThermoFisher) using UVITEC Alliance Q9. β-Actin was used as the loading control. Densitometric analysis was performed with Image J software. To measure released IL-1β, conditioned medium was collected as described above and IL-1β was measured through ELISA (BMS224-2, Invitrogen), according to manufacturer’s instructions. Cell number was used to normalise cytokine concentration.

The protein levels of TNF-α, IL-6 and IL-1β secreted via HRCECs was measured using specific enzyme-linked immunosorbent assay (ELISA) kits (BMS223HS, BMS213-2 and BMS224-2, Invitrogen, USA) according to the manufacturer's protocol.