Rs320 x ray machine

Senescence was induced in the cells by 20 Gy ionizing radiation using an RS320 X-Ray machine (X-Strahl, Camberley, United Kingdom) (Voskamp et al., 2021 (link)). MSCs in a monolayer were irradiated in a T175 flask (60%–70% confluency) for 22 min (20 Gy). Then, 24 h post-irradiation, the cells were trypsinized and seeded at a 9,600 cell/cm² density. Mock-irradiated MSCs were used as non-senescent controls and seeded at 2,300 cells/cm². Seven days post-irradiation, irradiated and non-irradiated MSCs were trypsinized, mixed (0, 25, 50, 75%, and 100% irradiated versus non-irradiated cells), and centrifuged at 300 g for 8 min to obtain pellets of 2 × 10⁵ cells. To induce chondrogenesis, cell pellets were cultured in a chondrogenic medium, containing a DMEM-HG medium (Invitrogen brand Thermo Fisher Scientific), supplemented with 1% ITS (BD, Franklin Lakes, NJ, United States), 1.5 μg/mL fungizone (Invitrogen brand Thermo Fisher Scientific), 50 μg/mL gentamicin (Invitrogen brand, Thermo Fisher Scientific), 1 mM sodium pyruvate (Invitrogen brand, Thermo Fisher Scientific), 40 μg/mL proline (Sigma-Aldrich), 10 ng/mL TGFβ1 (R&D Systems), 0.1 mM ascorbic acid-2-phosphate (Sigma-Aldrich), and 100 nM dexamethasone (Sigma-Aldrich) for 7, 14, or 21 days. The medium was renewed twice a week.