Lipid peroxidation malondialdehyde mda assay kit

Fisetin, glucose and dimethyl sulfoxide (DMSO) were obtained from Sigma–Aldrich (St. Louis, MO, United States). Fisetin (the purity is > 98%) was dissolved in < 0.1% of DMSO solution. LY294002 (the specific inhibitor of PI3K) was bought from MedChemExpress (Shanghai, China). Cell counting kit-8 (CCK-8) was purchased from Dojindo China Co., Ltd. (Shanghai, China). LDH cytotoxicity assay kit, lipid peroxidation [malondialdehyde (MDA)] assay kit, superoxide dismutase (SOD) assay kit, and Hoechst 33258 were obtained from Beyotime (Shanghai, China). Dulbecco’s modified eagle’s medium (DMEM) and phosphate-buffered saline (PBS) were purchased from Biological Industries (Shanghai, China). Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, United States). Antibody against PI3K (#4257), Phospho-PI3Kp85/p55 (#4228), Akt (#4691), Phospho-Akt (#4060), CREB (#4820), and p-CREB (#9198) were obtained from Cell Signaling Technology (Beverly, MA, United States). Antibody against β-actin was purchase from Absin (Shanghai, China).

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).

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.

Purified natural extracts of AA (97%) and dimethyl sulfoxide (DMSO) were purchased from Merck KGaA. SB203580 and SP600125 were purchased from Merck KGaA. Dulbecco's modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Thermo Fisher Scientific, Inc.). The enhanced chemiluminescence detection substrate and restore western blot stripping buffer were purchased from Thermo Fisher Scientific, Inc. The cell counting kit-8 (CCK-8) assay kit was obtained from Dojindo Molecular Technologies, Inc. The total SOD assay kit, lipid peroxidation malondialdehyde (MDA) assay kit, BCA protein assay kit, and mitochondrial membrane potential (∆Ψm MMP) assay kit were purchased from Beyotime Institute of Biotechnology. All antibodies were purchased from Cell Signalling Technology, Inc. Unless indicated otherwise, all other chemicals and materials were purchased from Merck KGaA.
AA solution was freshly prepared as a stock solution in DMSO and diluted using saline to a final concentration. The vehicle control was prepared by mixing DMSO with normal saline (0.1% v/v).

HT29, CT26, LOVO, MC38, and HCT116 cells were seeded in 96 well plate (5 × 10⁵ per well) overnight before CAP treatment. After 72 h of culture, cells were lysed using Western and immunoprecipitation lysis buffer 50 μL per well (Cat# P0013, Beyotime Biotechnology, Shanghai, China). The lysates were homogenized, and the resulting homogenates were centrifuged at 1,600xg at 4 °C for 10 min. The supernatants were collected and assessed using the Lipid Peroxidation Malondialdehyde (MDA) Assay Kit (Cat# S0131, Beyotime Biotechnology, Shanghai, China). Specifically, 200 µL of thiobarbituric acid (TBA) reagent was added to 100 µL of the cell suspension, and the mixture was heated in a boiling water bath for 15 min. After cooling and centrifuging the reaction mixture at 1,000xg for 10 min, the supernatant was separated, and absorbance was measured at 530 nm using the multi-mode microplate reader (SYNERGY H1, BioTek, Vermont, VT, USA). MDA levels were expressed as units (U) per 1 × 10⁶/mL cells and as nmol/mg protein.