Catalase cat assay kit

The brain tissues were collected and homogenized using 0.1 M PBS (pH 7.4), and the tissue homogenates were centrifuged at 3000 g for 20 min at 4°C. Lipid Peroxidation malondialdehyde (MDA) Assay kit (#S0131S), Catalase (CAT) Assay kit (#S0082), Total Glutathione Peroxidase (GPx) Assay kit (#S0058) and Nitric oxide (NO) Detection kit (#S0023; Beyotime, Shanghai, China) were used to determine the generation of MDA, CAT, GPx and NO according to the manufacturer’s instructions. To assess the inflammatory response, the levels of interleukin (IL)-6 (#PI328), tumor necrosis factor (TNF)-α (#PT516), IL-4 (#PI615), and IL-10 (#PI525) in tissue homogenates were measured using ELISA kits (Beyotime) according to the manufacturer’s protocols. Briefly, samples were added into a simpleStep ELISA plate that had been coated with monoclonal antibody specific for TNF-α, IL-6, IL-4, or IL-10 (from ELISA kits). Following incubation at 37°C for 30 min, the plates were washed 3 times with PBS. Then, samples were incubated with TNF-α, IL-6, IL-4, or IL-10 Detector antibodies (from ELISA kits) at 37°C for 1 h. Then 100 µl of the TMB substrate was added into each well and the absorbance at 405 nm was detected by an ELISA instrument (Thermo Fisher Scientific Inc.) after a 20-min incubation with the chromogenic agent.

H293T cell lines were purchased from the Shanghai Institute of Cell Biology (Shanghai, China). Dulbecco’s modified eagle’s medium-high glucose (DMEM), penicillin/streptomycin, trypsin, and phosphate buffered saline (PBS) were purchased from Wisent Biotechnology Co., Ltd. (Nanjing, China). Fetal bovine serum (FBS) was purchased from Gibco Inc. (Canyon, OR, USA). The bovine serum albumin (BSA) and bicinchoninic acid (BCA) protein assay kit were purchased from Solarbio Biotechnology Co., Ltd. (Beijing, China). Dimethyl sulfoxide (DMSO), radio immuno-precipitation assay (RIPA) buffer, phenylmethylsulfonyl fluoride (PMSF), total superoxide dismutase (SOD) assay kit with WST-8, catalase (CAT) assay kit, and reactive oxygen species (ROS) assay kit were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Cell counting kit-8 (CCK-8) was purchased from Tongren Chemical Research Institute (Kumamoto, Japan).

hDFSCs were cultured as above for 2–4 days before assays. According to a ROS assay kit (Beyotime, Shanghai, China), after washed three times with PBS, hDFSCs were incubated in 10 µM DCFH-DA solution at 37 °C for 20 min and observed by a laser confocal microscope (Zeiss, Oberkochen, Germany) at the wavelength of 488/525 (excitation/emission). For quantitation, hDFSCs were dissociated by 0.25% trypsin–EDTA and incubated as described above and the relative mean fluorescence intensity (MFI) was analyzed and normalized to the control using flow cytometry (BD, NY, USA) and FlowJo (BD, NY, USA). A superoxide assay kit (Beyotime, Shanghai, China), a hydrogen peroxide (H₂O₂) assay kit (Solarbio, Beijing, China), a total glutathione assay kit (Beyotime, Shanghai, China), a total superoxide dismutase (SOD) assay kit (Beyotime, Shanghai, China) and a catalase (CAT) assay kit (Beyotime, Shanghai, China) were used, respectively. Absorbance was measured by a microplate reader (Tecan, Männedorf, Switzerland). Protein concentration of each sample was determined at A280 using Nanodrop2000 (Thermo Scientific, MA, USA).

Collagenase II was obtained from Sigma-Aldrich (St Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM) with 100 U penicillin and 100 μg streptomycin and fetal bovine serum (FBS) were from Gibco™ (Grand Island, NY, USA). FITC Annexin V Apoptosis Detection Kit was from BD PharmingenTM (San Diego, CA, USA). Cell Counting Kit-8 (CCK-8), 0.25% trypsin, ROS assay kit, malondialdehyde (MDA) assay kit, catalase (CAT) assay kit, caspase-9 and caspase-3 colorimetric assay kits, cytochrome C ELISA kit, superoxide dismutase (SOD)-2 assay kit, ATP assay kit, and BCA Protein assay kit were purchased from Beyotime Biotechnology (Shanghai, China). TRIzol was from Invitrogen (Carlsbad, CA, USA). High Capacity cDNA Reverse Transcription kit was obtained from Applied Biosystems (Foster City, CA, USA). SYBR® Select Master Mix was obtained from Applied Biosystems (Austin, TX, USA). Collagenase II was dissolved in DMEM and diluted to 2 mg/mL to digest articular cartilage. Caspase inhibitor (Ac-DEVD-FMK) was purchased from Cell Signaling Technology (Danvers, MA, USA) and dissolved in dimethyl sulfoxide (DMSO). ASP was purchased from Shanghai Yilin Biotech. Co., Ltd (Shanghai, China). The purity of ASP is above 90%. The component sugars are glucose, galactose, arabinose, rhamnose, mannose, and xylose. The average molecular weight of ASP was 85.0 kDa.

β-asarone, Aβ_1–42, and 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma-Aldrich Inc. (St. Louis, MO, United States). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin were obtained from Hyclone (Thermo Fisher Scientific, WLM, Mass, United States). Phosphate-buffered saline (PBS) was obtained from Nanjing SunShine Biotechnology Co., Ltd. (Nanjing, China). A total superoxide dismutase (SOD) assay kit, lipid peroxidation malondialdehyde (MDA) assay kit, catalase (CAT) assay kit, total glutathione peroxidase (GSH-PX) assay kit, lactate dehydrogenase (LDH) cytotoxicity assay kit, and 2′, 7′-dichlorofluorescin diacetate (DCFH-DA) probe were purchased from Beyotime Biotechnology (Shanghai, China). An apoptosis detection kit was purchased from Thermo Fisher Scientific (WLM, Mass, United States). MitoSOX Red Mitochondrial Superoxide Indicator was purchased from Yeasen Biotech Co., Ltd. (Shanghai, China). A mitochondrial membrane potential assay kit with JC-1 was obtained from Solarbio (Beijing, China). Antibodies against HO-1 were obtained from Gene Tex Inc. (SA, Texas, United States). Antibodies against Nrf2, Bax, Bcl-2, cleaved caspase-3, P13K, P-P13K, P-Akt, Akt, β-actin, and Lamin A were obtained from Abcam (Cambridge, United Kingdom).

PD and its specific vehicle (ethanol 70%, propylene glycol 20%, and NaHCO₃ 10%) were supplied by Neptunus Co. (Shenzhen, Guangdong, China); its purity was over 99.5%. Antibodies against Bcl-2, Bax, SOD2, and acetylated SOD2 were obtained from Epitomics (Burlingame, CA, USA). The SOD activity assay kit was obtained from Dojindo Molecular Technology Inc. (Gaithersburg, MD, USA). Anti-SIRT1 and PGC-1α antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The SIRT1 activity assay kit was from Abcam (Cambridge, UK). The reduced glutathione/oxidized glutathione (GSH/GSSG) and catalase (CAT) assay kits were from Beyotime Biotech (Beijing, China). All other chemicals were from Sigma (St. Louis, MO, USA).