Mitochondrial ATP activity was assayed using an ATP bioluminescence assay kit (Beyotime Institute of Biotechnology, China), according to the manufacturer's instructions. Mitochondrial transmembrane potential (△Ψm) in HPMECs was measured as the manufacturer's direction. Briefly, 25 nmol/L TMRM dye (Molecular Probes, Invitrogen, USA) was added to ECM medium for 30 min and assessed via spectrophotometer (SpectraMax 5; Molecular Devices, Sunnyvale, CA, USA). HPMECs were treated with salmon sperm DNA (10 μg/mL) for 2 h and then mitochondrial ROS was detected using MitoSOX (Invitrogen, USA) according to the manufacturer's instructions and measured via spectrophotometer. MPO, MDA, SOD and total antioxidant capacity were measured by MPO assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), MDA assay kits (Beyotime Institute of Biotechnology, China), SOD assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and a rapid ABTS method (Beyotime Institute of Biotechnology, China), respectively.
Rapid abts method
Manufactured by Beyotime
Sourced in China
The Rapid ABTS method is a laboratory equipment used for the quantitative determination of antioxidant activity. It provides a fast and reliable way to measure the radical scavenging capacity of various samples.
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Lab products found in correlation
Total antioxidant capacity assay kit, by Beyotime (2 mentions)
Spectramax5, by Molecular Devices (1 mentions)
Sod assay kit, by Nanjing Jiancheng (1 mentions)
Mda assay kit, by Beyotime (1 mentions)
Mpo assay kit, by Nanjing Jiancheng (1 mentions)
Mitosox, by Thermo Fisher Scientific (1 mentions)
Atp bioluminescence assay kit, by Beyotime (1 mentions)
Dpph antioxidant assay kit, by Dojindo Laboratories (1 mentions)
3 protocols using rapid abts method
1
Mitochondrial Bioenergetics and Oxidative Stress Assay
2
ABTS Radical Scavenging Assay for Recombinant Trx-Like Protein
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The total antioxidant capacity assay kit, along with the rapid ABTS method (Beyotime, Shanghai, China), was used to determine the ABTS radical (ABTS+.) scavenging ability of refolded rPeTrxl, according to the manufacturer’s protocol. Briefly, different concentrations (0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL) of refolded rPeTrxl diluents were mixed with ABTS working fluid in a 96-well plate. PBS and different concentrations (0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL) of GSH were used as the negative and positive controls, respectively. The same volume of distilled water that replaced the reaction mixture was used as a blank control. Each concentration was measured in triplicate, and the Ab was monitored at 414 nm. The IC50 was calculated using SPSS 20.0, and all data are presented as the mean ± SD. The ABTS radical scavenging ability (%) of refolded rPeTrxl was calculated as follows:
3
Antioxidant Capacity Evaluation
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A total of 1 mg powder sample was dissolved in 2 mL of 50% methanol aqueous solution, and extracted for 10 min by ultrasound. Then, the antioxidant activity of powder, based on the scavenging activity of ABTS and DPPH free radicals, was carried out by Total Antioxidant Capacity Assay Kit with a Rapid ABTS method (S0121, beyotime) and DPPH Antioxidant Assay Kit (D678-12, dojindo), respectively. The antioxidant activity was expressed as millimole Trolox equivalent antioxidant capacity per kilogram powder sample (mmol TEAC/kg powder).
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