p65 immunofluorescence

Chondrocytes and synoviocytes were seeded in an 8 wells chamber slides at a density of 3 × 10⁴/cm² cells for 3 days in DMEM (SIGMA-ALDRICH) with 10% FCS. Successively, cells were treated with IL-1β (5 ng/mL, R&D) for 18 h, then the medium containing IL-1β was removed and substituted in half of the wells with fresh DMEM + 10% FBS (CTR) and in the other half with the same medium containing 10 μg/mL of sEV and cultured for 4 and 15 h. At the end of each experimental time point (4 h and 15 h) the cells were fixed with 4% PFA, treated for antigen unmasking with a solution of 0.02 U/mL of chondroitinase ABC in 50 mM TRIS–HCl pH 8 (for chondrocytes) or with a solution of 0.1% of Triton X-100/PBS (for synoviocytes) and blocked with 4% bovine serum albumin (BSA) (SIGMA-ALDRICH) in 0.1% Triton X-100/TBS (dilution buffer, used for primary and secondary antibodies dilution) to avoid unspecific bindings. Successively, cells were incubated with rabbit anti-human p65 (ABCAM AB7970, rabbit polyclonal antibody, 5 µg/ml) for 4 h at RT, followed by incubation with 15 μg/mL of donkey anti-rabbit IgG Alexa Fluor 555 (THERMOFISHER SCIENTIFIC). The nuclei were labeled with a DAPI solution (SIGMA-ALDRICH). Slides were mounted with the antifade reagent and examined under the NIKON A1-R confocal laser scanning microscope as described above.