Sa β gal activity

SA‐β‐gal is now a widely used biomarker in studies of cellular senescence because it is very easily detected and the activity is strongly associated with cellular senescence, although there is no specific marker to detect cellular senescence (Dimri et al., 1995). SA‐β‐gal activity was detected according to the manufacturer's protocol (Bio Vision, Milpitas, CA, USA).

The widely used biomarker for senescent cells is the SA-β-galactosidase activity. SA-β-gal activity is detectable at pH 6.0 in senescent cells (BioVision, Milpitas, CA). Beta-galactosidase activity is expressed from GLB1, the gene encoding lysosomal-galactosidase. Cardiomyocytes H9c2, endothelial cells EA.hy926, mouse embryonic fibroblasts (MEFs) and neonatal mouse cardiac fibroblasts (nMCFs) were cultured in 12-well culture plates and pretreated with TM5441 for 24 h followed by Doxorubicin (50 nM) treatment in triplicate for 4 days. At the end of treatment, cells were processed for SA-β-gal using Senescence Assay Kit (Catalog Cat#K320-250; BioVision, Milpitas, CA). In brief, culture medium was removed and cells were washed once with 1X PBS and fixed with fixative solution for 15 min at room temperature. After fixation, cells were washed twice with 1XPBS and staining solution containing staining supplement and X-gal was added to each well and mixed. Plates were covered with aluminum foil and incubated overnight at 37°C without CO₂. The SA-β-gal staining in cells were observed under a microscope for development of blue color and photographed.