Senescence β gal staining kit

Chondrocytes were stained using a Senescence β-gal Staining Kit (C0602, Beyotime, China) following the manufacturer’s instructions. Briefly, the cells were initially fixed and incubated with mixed staining solution in a CO₂-free dry incubator at 37°C for 12 h. Subsequently, positive (blue) cell ratios of five random fields were counted in all assays using a 100× magnification bright-field microscope [34 (link)]. For each group, three pictures were taken and the positive cells dyed blue were quantified by Image J software (National Institutes of Health, Bethesda, MD).

CNE2-EV&SOX1 and HONE1-EV&SOX1 cells were plated in a 6-well plate. The Senescence-β-Gal Staining Kit (Beyotime Institute of Biotechnology, Jiangsu, China) was used according to the manufacturer’s instructions. Briefly, cells were fixed with fixation buffer for 10 min after rinsing three times in PBS for 5 min at RT. The cells were stained with staining buffer at 37°C overnight covered with plastic wrap. The cells were rinsed three times in PBS for 5 min at RT and observed under a microscope.

VE, OA (> 98%), GA (> 98%), luteolin, vanillin, and Al(NO₃)₃·9H₂O were purchased from Aladdin Reagent Company (Shanghai, China). d-Gal and Folin–Ciocalteu were purchased from Beijing Solarbio Science and Technology Company (Beijing, China). Rutin (> 98%) was purchased from Guizhou Dida Technology Co., Ltd. (Guizhou, China). All other materials were the highest grade available.
Kits for T-AOC, SOD, MDA, CAT and AChE were purchased from the Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). A protein assay kit was bought from Bio-Rad Laboratories (Hercules, CA, USA), and the HE kit was purchased from Beijing Solarbio Science and Technology Company (Beijing, China). A senescence β-gal staining kit was obtained from the Beyotime Institute of Biotechnology (Haimen, China).

Senescence associated β-gal staining was performed using a senescence β-gal staining kit (C0602, Beyotime, China) according to the manufacturer's instructions. Briefly, the adherent cells were fixed in 4% paraformaldehyde and stained with β-gal staining solution at room temperature (RT) overnight.

The feature of cell senescence was evaluated by enhanced senescence associated β-galactosidase (SA-β-gal) activity (21 (link)). The detection of SA-β-gal activity was performed using senescence β-gal staining kit (Beyotime, China) according to the manufacturer's instruction. Briefly, cells were rinsed twice with phosphate-buffered saline (PBS), fixed with 2% formaldehyde/0.2% glutaraldehyde for 15 min at room temperature and incubated with SA-β-gal staining solution for 12 h at 37°C in humidified incubator without CO₂. The cells were observed under fluorescence microscope (MF52, Mshot, China).