Malondialdehyde mda assay kit

Trypsin from bovine pancreas (≥2,500 IU/mg) and methyl viologen dichloride were purchased from Aladdin Co. (Shanghai, China). Papain (6,000 USP U/mg) was purchased from Solarbio Co. (Beijing, China). 2,7-Dichlorofluorescein diacetate (DCFH-DA) and 5-fluoro-2′-deoxyuridine (FUdR) were bought from Sigma-Aldrich Co. (St. Louis, MO, USA). Sephadex G-25 (medium) was obtained from GE Healthcare Co. (Uppsala, Sweden). Ammonium sulfate (analytical grade) was obtained from Guangzhou Chemical Reagent Factory (Guangzhou, China). SOD, CAT, and malondialdehyde (MDA) assay kits were purchased from Beyotime (Haimen, China). BCA Protein Assay Kit was bought from Pierce Chemical Co. (Rockford, IL, USA).

Bicinchoninic acid (BCA) protein assay kit (#MA0082-1) and RIPA lysis buffer (#MA0153) were purchased from Meilunbio (Dalian, China). L-arginine (L-Arg) monohydrochloride (#A6969), Collagenase IV (#C9407), Disulfiram (#HY-B0240) and DAPI (#D9542) were from Sigma-Aldrich (St. Louis, MO, USA) of Merck. GSDMD antibody (#AF4012) was from Affinity (Shanghai, China), REDD1 (Regulated in Development and DNA Damage Responses 1). Antibody (#sc-376671) and p-NF-Kb (p-P65) (#sc-398442) were from Santa Cruz Biotechnology (CA, USA). Resveratrol (#A2122398) was from Aladdin (Shanghai, China). TXNIP(#ab188865) and HIF-1α (#ab1) were from Abcam (Boston, MA, USA). Malondialdehyde (MDA) assay kit (#S0131S), catalase (CAT) (#S0051) assay kit, total glutathione peroxidase (GPX) assay kit with NADPH (#S0058) and anti-β-actin antibody (AF0003) were purchased from Beyotime Biotech (Shanghai, China). Mouse IL-1β ELISA kit (E-EL-M0037c) was purchased from Elabscience Biotechnology Co. Ltd. (Wuhan, China). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) of Merck.

Polygonatum sibiricum polysaccharides (PSP) were purchased from YuanYe Bio-Technology Co., Ltd. (Shanghai, China) with a purity of 70%. D-gal (purity > 98%) was purchased from Nanjiang Pharmaceutical Co., Ltd. (Kunming, China). Aging β-galactosidase staining kit, total superoxide dismutase (SOD) assay kit, malondialdehyde (MDA) assay kit, and reactive oxygen species (ROS) assay kit were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). The antibodies were purchased from Abcam (Cambridge, USA); EasySee Western Blotting Kit was purchased from Beijing TransGen Biotech Co., Ltd. (Beijing, China).

Sea Buckthorn proanthocyanidins (SBP, Purity: 91.5%) were provided by Puredia Limited (Qinghai, China). The SBP was extracted from Sea buckthorn by water extraction and macroporous resin column chromatography (MRCC), trademarked as CyanthOx™. HSFs (derived from human superficial skin tissue), fetal bovine serum (FBS), Dulbecco’s modified Eagle medium (DMEM), phosphate-buffered saline (PBS), penicillin–streptomycin solution (double antibody) 100×, and 0.25% trypsin solution were purchased from Xiamen Immocell Biotechnology Co., Ltd. (Xiamen, China). A Cell Counting Kit 8 (CCK-8) ROS detection kit was purchased from Beijing Solar Science & Technology Co., Ltd. (Beijing, China). A senescence β-galactosidase staining kit, SOD assay kit and WST-8, GSH assay kit, malondialdehyde (MDA) assay kit, and particular fixative solution, washing solution, blocking solution, Alexa Fluor 488-labeled Goat Anti-Rabbit IgG(H+L), and antifade mounting medium with DAPI for immunofluorescence staining were all purchased from Beyotime Inc. (Shanghai, China). Primary antibodies, including anti-TGF-β1, anti-Smad 3, anti-phospho-Smad 3 (Ser425), anti-Smad 4, anti-type I collagen, and β-actin, were obtained from Affinity Biosciences (OH, USA). MMP-1, MMP-3, and TIMP-1 Elisa assay kits were obtained from Elabscience Biotechnology Co., Ltd. (Wuhan, China).

Mannitol (CAS:69–65-8) and MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) kit were purchased from Aladdin (Shanghai, China), Dulbecco’s modified Eagle’s medium: nutrient mixture F-12 (DMEM-F12) was from Gibco (Invitrogen, Carlsbad, CA, USA), new-born calf serum (NCS) was from Tianhang (Hangzhou, China). The bicinchoninic acid (BCA) protein assay kit, 4',6-diamidino-2-phenylindole (DAPI) staining solution, malondialdehyde (MDA) assay kit were obtained from Beyotime Institute of Biotechnology, and glutathione (GSH) assay kit was purchased from Nanjing Jiancheng Bioengineering Institute, Nanjing, China. The Annexin V-FITC apoptosis detection kit was from Nanjing KeyGen Biotech Co. Ltd Nanjing, China. Trypsin-EDTA, Triton X-100, bovine serum albumin (BSA) were from Amresco (Solon, OH, USA). FITC-Phalloidin was obtained from Sigma (Chemical Co., St. Louis, MO, USA).

As described, dECM hydrogels were added to 24 well plates at 200 µl/well and incubated at 37°C for 30 min. RAW264.7 cells were collected and rinsed in PBS. WST-8/enzyme working solutions were added to the cells by the instructions of the superoxide dismutase (SOD) assay kit (Beyotime Biotechnology, No. S0103). Cells were incubated at 37°C for 30 min and centrifuged at 1000 g for 10 min. The 96-well plates were then plated with 200 μl of the supernatant. Absorbance was measured at 450 nm using a full-wavelength microplate reader (Thermo Fisher Scientific). Treated RAW264.7 cells were lysed for 30 min and analyzed according to the instructions of the malondialdehyde (MDA) assay kit (Beyotime Biotechnology, no. S0103). MDA assay solution was heated at 100°C for 15 min, cooled in a water bath to room temperature, centrifuged at 1000 g for 10 min, and 200 μl supernatant was added to 96-well plates. Complete wavelength absorption was measured at 532 nm. The assays were repeated three times under the same conditions.