Gpx cellular activity assay kit cgp 1

SOD enzymatic activity was determined using a spectrophotometric SOD Assay Kit (Sigma, Darmstad, Germany) according to the manufacturer’s instructions. The indirect measurement of SOD activity was obtained based on the water soluble tetrazolium (WST) salt reaction with superoxide anion producing a water-soluble formazan dye.
GPx enzyme activity was assessed using the GPx Cellular Activity Assay Kit CGP-1 (Sigma). The indirect determination method of the GPx activity is based on the nicotinamide adenine dinucleotide phosphate (NAPDH) concentration decrease in the reaction media during which NADPH is oxidized to NADP+.
Lipid peroxidation as reflected by malondialdehyde (MDA) levels was assessed using thiobarbituric acid-reactive substances (TBARs) determination method [30 (link)]. Trichloroacetic acid (50%, 0.25 mL), thiobarbituric acid (0.73%, 0.255 mL) and tissue extracts (0.05 mL) were mixed and vortexed. Afterwards, a 20 min incubation at 100 °C (boiling water bath), and a 10 min centrifugation (3000 rpm) were performed. The supernatants were exposed to a 532 nm spectrometry system and the absorbance was read against MDA standard curve (the results were expressed as mmol MDA/mL tissue extract).

To carry out our activities, we used analytical grade solvents and various classic reagents. Ethyl acetate and 2-thiobarbituric acid were purchased from Sigma-Aldrich (Steinheim, Germany); potassium persulfate, 2,2′ azino-bis(3-ethylbenzothiazoline 6-sulphonate), and trichloroacetic acid were supplied by Fluka Chemie (Buchs, Switzerland); dichloromethane, ferric dichloride, and ethanol were sourced from Probalo (Paris, France); butanol was sourced from SDS (Peyin, France). For cerebral tissue oxidative stress biomarkers, we used the spectrophotometric SOD Assay Kit (Fluka Chemie), GPX cellular activity assay kit CGP-1 (Sigma Chemicals), trichloroacetic acid 50%, and thiobarbituric acid 0.73%.