Cellular senescence β galactosidase staining kit

SA‐β‐gal staining was performed using the cellular senescence β‐galactosidase staining kit (Beyotime, C0602) in accordance with the instructions provided. After washing the cells with PBS and subsequent fixation, staining solution was added, followed by sealing with parafilm and overnight incubation at 37°C. Then, the cell climbing slices were removed and imaged with an upright optical microscope.

Anhydrous copper chloride (CuCl₂), 3,3’,5,5’-tetramethylbenzidine (TMB), bovine serum albumin (BSA), and sodium sulfide nonahydrate (Na₂S·9H₂O) were from Macklin Biochemical (Shanghai, China). Doxorubicin (Dox), N-(3-Dimethylaminopropyl)-N’-ethylcabodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were from Sigma-Aldrich (USA). Antibodies against p16^ink4a (polyclonal, ab108349), B2M (monoclonal, ab75853), high mobility group protein 1 (HMGB-1) (ab228624), matrix metalloprotein 13 (MMP-13) (ab39012), and type II collagen (Col-2) (ab34712) were from Abcam (UK). The cellular senescence β-galactosidase staining kit and cell counting kit (CCK-8) were from Beyotime Biotechnology (Shanghai, China). FITC-xtra and MitoROS™ 580 were from ATT Bioquest (USA). The Evo M-MLV Reverse Transcription Reagent and SYBR Green Pro Taq HS qPCR Kit were from Accurate Biology (China). The chondrogenic differentiation kit was from Cyagen Biosciences (USA).

Tibiae and femurs of wild-type and p27^-/- mice were removed under aseptic conditions, and bone marrow cells were flushed out with DMEM containing 10% FCS, 50 µg/mL ascorbic acid, 10 mM β-glycerophosphate, and 10^-8 M dexamethasone. Cells were dispersed by repeated pipetting, and a single-cell suspension was achieved by forcefully expelling the cells through a 22-gauge syringe needle. Total bone marrow cells were cultured in six-well-plates at 10⁶ cells/well in 2 mL of the above-mentioned medium in the absence or presence of Shh-N (200 ng/ml) or Shh antagonist KAAD-Cyclopamine (KAAD,2.5μM). Medium was changed every 3 days, and cultures were maintained for 10 to 14 days. At the end of the culture period, cells were washed with PBS, fixed with PLP fixative, and stained cytochemically for ALP as described previously 25 (link) to determine ALP-positive CFU-f (CFU-fap). After petri dishes were dried and imaged, they were stained with methyl blue to determine total CFU-f. Cellular senescence staining was performed with a cellular senescence β-galactosidase staining kit (# C0602, Beyotime Biotechnology, Wuhan, China) according to the manufacturer's protocol.