Senescence β galactosidase staining kit

BMSCs were isolated as previously described (Li et al., 2015 (link); Yu et al., 2018 (link)). The isolated BMSCs were cultured with α-MEM supplemented with 15% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin in a humidified atmosphere of 5% CO2 at 37 °C to reach 80% confluence. Then the first-passage BMSCs were harvested and seeded in culture dishes for enrichment of cell populations. When the second-passage reach confluence after 1–2 week, they were subcultured. Only third-passage BMSCs were applied to perform further study unless specified otherwise.
The senescent BMSCs were stained by a senescence β-galactosidase staining Kit (Solarbio Science & Technology) according to the manufacturer’s instructions. Briefly, after washing with PBS, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Then the cells were stained with working solution overnight at 37 °C. Five different fields were randomly selected under a microscope to count the SA-βGal-positive (blue cells) and the percentage of SA-βGal-positive were calculated.

The activity of SA-β-gal was performed by a senescence β-galactosidase staining kit following the manufacturer’s instructions (Solarbio, Beijing). Briefly, the treated cells on 24-well chamber slides and lung tissues were fixed with 4% formaldehyde for 15 min at room temperature, rinsed three times with PBS for 3 min, and then incubated with freshly prepared SA-β-Gal staining solution at 37 °C overnight. Slides were rinsed twice with PBS for 1 min at room temperature. The positive cells were observed and imaged using an electron microscope.

BMSCs with Asph siRNA interference were detected by senescence β-Galactosidase staining kit according to the manufacturer’s instructions (G1580, Solarbio Life Science). Briefly, we firstly prepared the staining working solution which comprised of 10 μl β -galactosidase stain A, 10 μl β -galactosidase stain B, 930 μl β -galactosidase stain C and 50 μl X-Gal solution for each well. Then, we rinsed the cell plates with PBS followed by the fixation by 1 ml fix solution of β-galactosidase staining at room temperature for 15 min. After removal of fix solution, the plates were rinsed for three times. Then, cells were incubated with 1 ml staining working solution in incubator at 37°C overnight. Finally, the plates were visualized through Olympus microscope.