F9911

One hundred μl each of recombinant fibrinogen (F3879, sigma), full-length fibronectin (F2006, Sigma), HBD (F9911, Sigma) and CBD (F0162, 45-kDa, Sigma) domains of fibronectin at a concentration of 5 μg/ml in 50mM Na₂CO₃, pH 9.6 was coated onto ELISA plates (IWAKI) at 4 °C overnight. After three-time washes with PBS, the plate was blocked with 1% (w/v) BSA in 50 mM Na₂CO₃ (pH 9.6) and incubated at room temperature for 1h. The ELISA plate was then washed three times and then incubated with 3xFlag-tagged, recombinant VpadF truncation fragments at room temperature for 1 h. After triple washes, 100 μl mouse α-3xFlag-HRP antibody (diluted 1:1000 0) was added to each well and incubated at room temperature for 1 h. 100 μl TMB (T4444, sigma) was added to each well for color development (5 min, room temperature). After quenching with 100 μl H₂SO₄ (1 M), absorbance was read at 450 nm. BSA was coated simultaneously as negative control.

For the stimulation experiments, IVD cells were cultured for 24 h in a 6-well plate at a density of 2 × 10⁵ cells/well in DMEM/F12 supplemented with 1% Antibiotic-Antimycotic and 10% FCS. On the next day, cells were serum starved in media supplemented with Polymyxin B (100 units/mL, 81271, Sigma-Aldrich) with or without Sparstolonin B (SsnB, 25 μM, TRL-2/TRL-4 inhibitor, SML1767, Sigma-Aldrich) for 2 h prior to stimulation with FnF. Afterwards, cells were treated with 30 kDa proteolytic fragments from human plasma fibronectin (FnF, 500 nmol/L, F9911, Sigma-Aldrich) or recombinant canine IL-1β, which was used as positive inflammation control (10 ng/ml, 3747-CL-025, R&D Systems, Inc., Minneapolis, MN 55413, USA) for 18h. Untreated NP cells served as a negative control group, NP cells treated with Sparstolonin B alone were used as a control to the combination treatment of FnF, and Sparstolonin B. Used concentrations were based on the range of FnF found in IVDs during degeneration (19 (link), 29 (link)). After the incubation time, media supernatants were collected, and cells were lysed using RLT Lysis Buffer (79,216, Qiagen, Hilden, Germany). Both cell lysates and supernatants were stored at −80°C until further analysis.