Catalase activity assay kit

Primary antibodies we used were anti-H. pylori (ab7788, Abcam), anti-HSPA8 (10654-1-AP, Proteintech), anti-HSPA1A (10995-1-AP, Proteintech), anti-HSPA1B (25405-1-AP, Proteintech), anti-BRCA1 (22362-1-AP, Proteintech), anti-SUZ12 (20366-1-AP, Proteintech), anti-FANCM (12954-1-AP, Proteintech), anti-ZO-1 (21773-1-AP, Proteintech), anti-Claudin 1 (13050-1-AP, Proteintech), anti-Occludin (66378-1-Ig, Proteintech), anti-β-Actin (4967S, Cell Signaling Technology), and anti-GAPDH (2118S, Cell Signaling Technology). Secondary antibodies used included anti-mouse conjugated to horseradish peroxidase (A2304, Sigma-Aldrich) and anti-rabbit conjugated to horseradish peroxidase (NA9340, GE Healthcare) for western blotting; anti-rabbit conjugated to Alexa Fluor 568 (A11011) and anti-mouse conjugated to Alexa Fluor 488 (A21202) were from Life Technology for confocal microscopy.
The Catalase Activity Assay Kit was purchased from Abcam (ab83464). DSS (160110) was from MP Biomedicals (Shanghai) Co. Ltd. MitoSox Red mitochondrial superoxide indicator was from Thermo Fisher Scientific (M3608). The 8-OHdG ELISA Kit was from Abcam (ab201734).

Total antioxidant activity in protein lysates was measured following published methodology^{31 (link),32 (link)}. Briefly, protein lysates standardised to a concentration of 900 µg/mL or trolox standards were added to oxidised 2,2’-Azino-di-[3-ethylbenzthiazoline sulphonate] and reduction spectrophotometrically measured. Results are expressed relative to trolox activity (mmol/L trolox equivalent). Superoxide dismutase activity was determined in protein lysates using the Superoxide Dismutase Assay Kit (Cayman Chemical, Australia) following the manufacturer’s protocol. Catalase activity was determined in protein lysates using the Catalase Activity Assay Kit (Abcam, Australia) following the manufacture’s protocol. Glutathione peroxidase (GPx) activity was determined in protein lysates following published methodology^{33 (link)}.

After the cells had been treated with NPs in accordance with the instructor’s procedure, the SOD assay commercial kit (Superoxide Dismutase Activity Assay kit, Colorimetric, cat no = ab65354, from Abcam, Cambridge, UK) was used to measure SOD activity. The samples’ absorbance was determined at 450 nm with the help of a plate reader. Following the kit instructions, the CAT assay kit was utilized to investigate the activity of the CAT enzyme. Based on the breakdown of hydrogen peroxide, the enzyme is measured with the kit (Catalase Activity Assay Kit, Colorimetric/Fluorometric cat no = ab83464, from Abcam, Cambridge, UK). The levels of MDA activity were also measured using a kit. At 450 nm, the samples’ absorbance was measured. MDA and thiobarbituric acid combine to produce a complex, after which the absorbance was measured at 532 nm to assess lipid peroxidation (Lipid Peroxidation (MDA) Assay Kit (Colorimetric) cat no = ab233471, from Abcam, Cambridge, UK). The levels of GSH activity were also measured using a kit. Using a kinetic assay, the amount of GSH is measured by continuously reducing 5,5′-dithiobis (2-nitrobenzoic acid) in the presence of catalytic quantities (nmoles) of GSH (GSH Assay Kit, Colorimetric, cat no = ab239709, from Abcam, Cambridge, UK).

The activity of Catalase in cardiomyocytes was monitored with Catalase Activity Assay Kit (Abcam, # ab83464).

Liver, WAT, and BAT tissues were homogenized in ice-cold PBS. The sample were centrifuged at 4 °C at 10,000 × g and supernatant was collected and catalase activity was measured using the catalase activity assay kit (abcam, # ab83464).