Cell senescence β galactosidase staining kit

Senescence-associated SA-β-gal staining was performed according to the method described previously^{83 (link)}. Briefly, IMR90 cells were fixed in 2% formaldehyde and 0.2% glutaraldehyde at room temperature for 10 min, and stained in freshly prepared staining solution (all reagents were from Cell Senescence β-galactosidase Staining Kit, Beyotime, Haimen) at 37 °C overnight. Images were taken and the percentages of SA-β-gal-positive cells were counted and determined using Image J. Each group had three biological replicates.

The cell senescence β-galactosidase staining kit (Beyotime Biotechnology, Shanghai, China) was used to detect the level of senescence-associated β-galactosidase activity in hypoxia-induced PASMC proliferation. SA-β-Gal staining was performed according to the manufacturer’s instructions. Under an optical microscope, it can be observed that senescence cells are dyed into blue in hypoxia-induced PASMC proliferation. Briefly, cells were placed in a 3% small hypoxia compartment for 48 h. Next, cells were washed twice in phosphate-buffered saline (PBS); staining solution was added to the PASMCs for 15 min at room temperature. Then cells were washed with PBS for three times and stained with working solution overnight at a 37°C electrothermal incubator.

The SA-β-Gal staining was conducted employing the cell senescence β-galactosidase staining kit (Beyotime Biotechnology, China), based on the guidelines provided by the manufacturer. In a concise manner, the cells underwent a washing process utilizing PBS and were subsequently fixed with a solution comprising 2% paraformaldehyde and 0.2% glutaraldehyde for 5 min. Then, the cells underwent a washing procedure and were exposed to a staining solution containing SA-β-Gal for 16 h at 37 °C. Following the incubation, the cells underwent a process of washing, and a Nikon Eclipse Ni-U microscope was utilized to image the cells.

The RGC-5 cells were plated and cultured for a set duration in six-well plates. When the cells reached a confluence rate of 80%, the experimental groups were subjected to treatment with SPAM1 (1 µM–100 µM) for 24 h. The same volume of fetal bovine serum-free basal medium was administered to the control group without any treatment. The cell culture medium was aspirated, washed once with PBS, and then fixed and stained according to the instructions of the cell senescence β-galactosidase staining kit (Beyotime, Shanghai, China). After incubation overnight at 37 °C, the percentage of stained senescent cells was obtained by observing and counting under an ordinary light microscope. All experiments were performed with at least three parallel replicates and repeated three times.

SA-β-gal staining was performed using the Cell Senescence β-galactosidase Staining kit (Cat# C0602, Beyotime) according to the manufacturer’s instructions. Images were taken with an inverted light microscope (ECHO).