Sample activation kit 1

After peripheral blood drawing, each sample was centrifuged at 3500 rpm within 15 min from collection and stored at − 80 °C until analyzed. ELISA kits for the detection of human IL-6, IL-12p70, and TGFβ were obtained from Bio-Techne (UK). The IL-10 detection was assessed with High Sensitivity (HS) ELISA (Bio-Techne, UK). Serum cytokine’s levels were determined according to the manufacturer’s instructions. The IL-6 (sensitivity: 0.7 pg/ml), IL-10 HS (sensitivity: 0.17 pg/ml), IL-12p70 (sensitivity: 5 pg/ml), and TGF-β (sensitivity: 15.4 pg/ml) levels were calculated according to the specific standard curves. TGF-β measurement was performed after its activation (BioTechne, UK). Briefly, latent TGF-β1 serum was activated to its immunoreactive form, using solutions for acid activation and neutralization (Sample Activation Kit 1, R&D Systems®).

For some proteins, the amounts released from cartilage tissues were quantified by bead-based suspension array technology on a Luminex 100/200 analyzer (Luminex, Austin, TX, USA) using commercially available kits (Magnetic Luminex Performance Assay and Human Luminex Discovery Assay, R&D Systems, and C3a Human ProcartaPlex Simplex kit, ThermoFisher Scientific), according to the manufacturers’ instructions. Briefly, 50 µl of PBS containing the released proteins was incubated with a suspension of capture antibody-conjugated magnetic microspheres. The microspheres were then incubated with a biotinylated detection antibody and streptavidin–phycoerythrin conjugate, and fluorescence intensity was measured. For the measurement of TGF-β1, β2 and β3, samples were acid-activated prior to measurement using Sample Activation Kit 1 (R&D Systems).

The expression level of TGFβ2 released in the cell culture medium was detected by ELISA using the Human TGF-beta 2 DuoSet ELISA (R&D System, cat no. DY302, Minneapolis, MN, USA), DuoSet Ancillary Reagent Kit 2 (5 plates, R&D Systems, cat no. DY008, Minneapolis, MN, USA) and Sample Activation Kit 1 (R&D Systems, cat no. DY010, Minneapolis, MN, USA). For IL6 quantification from cell culture, ELISA was performed using the IL6 DuoSet ELISA Kit (R&D System, cat no. DY206-05, Minneapolis, MN, USA) along with the DuoSet Ancillary Reagent Kit 2 (5 plates, R&D Systems, cat no. DY008, Minneapolis, MN, USA).

The amounts of TNF-α, IL-1β, IL-6, IL-12 p40, and IL-10 in the culture supernatants were measured using commercial ELISA kits (BD Biosciences, San Jose, CA, USA). The amount of TGF-β was measured using an ELISA kit of R&D systems (Abingdon, UK) after treating the culture supernatant with Sample Activation Kit 1 (R&D systems) as suggested by the supplier.