Detection of senescence-associated β-galactosidase activity

Senescence-associated β-galactosidase (SA-β-gal) assay was performed as described previously [22]. Briefly, the SA-β-gal activity was measured using flow cytometric fluorescence detection in vitro. The HTFs were incubated with 5-Dodecanoylaminofluorescein Di-β-D-Galactopyranoside (C12FDG; Thermo Fisher, Eugene, OR, USA), a β-galactosidase substrate that becomes fluorescent after cleavage by the SA-β-gal. The SA-β-gal-positive cells and median fluorescence intensities (MFIs) were detected and quantified through flow cytometry. The MFIs and percentage of SA-β-gal-positive cells were measured separately. β-Galactosidase is a collective name for enzymes that cleave nonreducing β-D-galactose residues from glycoproteins, sphingolipids, and keratan sulfate in β-D-galactosides [23]. SA-β-gal activity can also be cytochemically detected using 5-bromo-4-chloro-3-indolyl-b-D-galactoside (X-gal; Sigma-Aldrich, St. Louis, MO, USA) as a substrate. In cytochemical or histochemical detection of SA-β-gal activity, the HTFs or frozen tissue sections (6 μm thick) were incubated with SA-β-Gal solution (X-gal, 1 mg/mL; citric acid/sodium phosphate, pH 5.8, 40 mM; potassium ferrocyanide, 5 mM; potassium ferricyanide, 5 mM; NaCl, 150 mM; MgCl2, 2 mM) for 12 h at 37°C. After PBS washing, SA-β-gal-positive cells were analyzed under light microscopy.