5 ethynyl 2 deoxyuridine edu staining kit

This study was approved by the Animal Ethics Committee of Xuzhou Medical University. SD rats (1-3 days old) were purchased from the Laboratory Animal Center (Xuzhou Medical University). The animals used in this study received humane care and handling. RSV and type I collagenase were obtained from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human TGF-β1 was purchased from Peprotech (Suzhou, China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Life Technologies (Grand Island, NY, USA). Cell Counting Kit-8 (CCK-8), 0.25% trypsin, dimethyl sulfoxide (DMSO), RIPA lysis buffer, phenylmethylsulfonyl fluoride (PMSF), and the bicinchoninic acid (BCA) protein assay kit were purchased from Beyotime (Guangzhou, China). The EdU (5-ethynyl-2-deoxyuridine) staining kit was purchased from RiboBio (Guangzhou, China). The hydroxyproline assay kit was purchased from Jiancheng Bioengineering Institute (Nanjing, China). A polymerase chain reaction (PCR) kit was purchased from Tiangen (Beijing, China). The mouse monoclonal antibody against Smad7 was purchased from Santa Cruz Biotechnology, Inc. (Paso Robles, CA, USA). The rabbit polyclonal antibody against GAPDH was purchased from Proteintech Group, Inc. (Rosemont, IL 60018, USA). Secondary antibodies conjugated to HRP were purchased from Zhongshan Jinqiao Biotechnology (Beijing, China).

Since senescent cells undergo cell cycle arrest, the defect of proliferation ability suggests cellular senescence. Thus, the proliferation ability of HUVECs was determined using the EdU (5-ethynyl-2′-deoxyuridine) staining kit (RiboBio Co., Ltd., Guangzhou, China) according to the protocol. Images were acquired by cell auto imaging system (EVOS FL Auto, Life Technologies, New York, USA). The EdU-positive cells were shown red. Ratio of proliferating cells was normalized to the total cell numbers stained with Hoechst (blue).