β galactosidase sa β gal staining kit

Senescence was detected by staining MSCs with a β-galactosidase (SA-β-gal) staining kit (Cell Signaling Technologies, Danvers, MA, USA) according to the manufacturer’s instructions, and analyzed with a direct-light microscope. Briefly, 1 × 10⁴ cells were plated in a 35-mm² dish and incubated overnight at 37°C. After removing the growth medium, cells were washed twice with PBS, fixed for 10–15 min at room temperature, and incubated at 37°C overnight in a dry incubator (atmospheric CO₂) with fresh β-gal staining solution. β-gal–positive cells were monitored under a microscope for the development of blue color and subsequently imaged.

The presence of senescent cells in skeletal muscle tissue [n = 8 for WT or mdx/utr(−/−) mice] and cells [n = 3 for WT or mdx/utr(−/−) cells, with 2 replicates for each cell population] cultured in vitro was individually measured using the regular senescence-associated β-Galactosidase (SA-β-gal) Staining Kit (Cell Signaling Technology, Danvers, MA, USA) and CellEvent Senescence Green Detection Kit (fluorescent) (Thermo Fisher Scientific), following the manufacturers’ protocol. The number of cells positive for β-gal activity at pH 6, a known characteristic of senescent cells, was determined.

NB-MSC senescence was assessed by staining with the β-galactosidase (SA-β-gal) staining kit (Cell Signaling Technology, Danvers, MA), according to the manufacturer’s instructions, and evaluated by direct-light microscopy.