Senescence associated galactosidase staining kit

The protocol was referred to the instruction of the senescence‐associated ß‐galactosidase staining kit (Beyotime Biotechnology). Briefly, a suitable amount of SA‐ß‐gal staining stationary solution was added into the cells treated by PBS or MA and then fixed at room temperature for 15 minutes. After washing with PBS for three times, a sufficient amount of pre‐prepared dyeing working liquid was added and incubated overnight in the incubator without CO₂ at 37°C. The working fluid was removed in the next day, washed with PBS and observed under a microscope (Nikon, ECLIPSE TE2000‐U). The blue‐stained cells were identified as senescent cells and were counted in ten fields of view randomly selected in each section to determine the percentage of ß‐galactosidase‐positive cells (magnification, ×200).

Senescence associated ß-galactosidase (SA-β-Gal) staining of Y-BMSCs and O-BMSCs cultured with/without OI-exo was performed using the senescence-associated ß-galactosidase staining kit (Beyotime, Jiangsu, China) according to the manufacturer’s instructions. After cell confluence, cells were fixed for 15 min and incubated overnight at 37°C with ß-Gal Staining Solution, then ice-cold PBS was used to quench the reaction. The staining was observed in bright field using an inverted optical microscope (Leica DMI6000B, Germany).