Senescence β-Galactosidase Staining Kit - Cell Signaling

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	Fix cells with with 2% formaldehyde/0.2% glutaraldehyde. Incubate for 12 hours at 37 ° C without Co2

Protocol tips
Fix cells with with 2% formaldehyde/0.2% glutaraldehyde. Incubate for 12 hours at 37 ° C without Co2

Publication protocol

For colony formation assay, 1000, 5000, or 10000 cells were plated in 35-mm dish, cultured for 7–10 days, and stained with crystal violet (Wako). Detection of SA-β-Gal activity was performed using Senescence β-Galactosidase staining kit (Cell Signaling Technology) according to the manufacturer’s instruction. Briefly, the cells were fixed with 2% formaldehyde/0.2% glutaraldehyde. After incubation with SA-β-Gal staining solution (1 mg/ml 5- bromo-4-chloro-3-indolyl-β-D-galactoside, 40 mM citric acid/sodium phosphate [pH 6.0], 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl2) for 12 h (HepG2 cells) or 24 h (U2OS and Hs68 cells), the cells were examined under fluorescence microscope (model BZ-8000; Keyence). At least 100 cells were counted to determine the percentage of SA-β-Gal positive cells. For BrdU incorporation assay, cells were labeled with 10 μM BrdU (Sigma) for 6 h (HepG2 cells) or 24 h (Hs68 cells). For BrdU immunostaining, the labeled cells were fixed with 3.7% formaldehyde in PBS and permeabilized with 0.5% TritonX-100. DNA was hydrolyzed by exposing cells to 2 N HCl for 10 min, and then cells were incubated with anti-BrdU antibody (BD Pharmingen, 555627) in Can Get Signal immunostain Solution B (TOYOBO) overnight at 4 °C followed by incubation with TRITC-conjugated secondary antibodies (Molecular Probes, T2762) for 1 h at room temperature. After staining nuclei with 10 μM Hoechst 33342, cells were examined under fluorescence microscope (model BZ-9000; Keyence).

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