Sod assay kit wst

IPA treated or not with 5-HT were homogenized in HEPES buffer containing (in mM): 20 HEPES, 1 EGTA, 210 mannitol, 70 sucrose, pH 7.2 with NaOH. Homogenized samples were centrifuged (1500 g, 5 min at 4°C) and supernatants were used for the determination of superoxide dismutase (SOD) activity. SOD enzyme activity was then determined as described in the procedure of the SOD Assay Kit-WST from Sigma and as carried out in previous studies (Peskin and Winterbourn, 2000 (link)). Briefly, the method is based on the inhibition of the SOD activity in presence of the highly water-soluble tetrazolium salt WST-1 [2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] that produces a colored water-soluble formazan dye (WST1-formazan) upon reduction with ${{\text{O}}_2}^{\underset{\_}{•}}$ . Levels of SOD activity were determined by measuring the absorbance of the WST1-formazan dye at 450 nm with a microplate reader (EL808, Bio-Tek instruments). SOD standard solutions ranging from 0.001 to 200 Units/ml were used to perform a calibration curve and SOD activity value was read from this curve and expressed as Units/ml for each sample. Tissue protein content of each sample was also quantified in mg/ml to normalize SOD enzyme activity, expressed as Units/mg of proteins.

Superoxide anion scavenging /superoxide dismutase (SOD) activity was determined using SOD assay Kit—WST (Sigma-Aldrich), a commercially available colorimetric microtiter plate method, according to the protocol given by the manufacturer. SOD activity of the extract was determined colorimetrically at 450 nm as the reduction of the Dojindo's highly water-soluble tetrazolium salt, WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) by superoxide anion, ${\text{O}}_2^{-}$ . Sample extract solution (20 μL) at different concentrations were added to the 96-well-plate, respectively. The reaction solutions were added as described in the protocol and then the plate was incubated at 37°C for 20 min prior to measurement of absorbance at 450 nm using a microplate reader. The percentage of SOD activity (percentage of WST-1 reduction) was calculated as follows (Tan et al., 2015 (link)):

Newly eclosed male flies were pretreated with KRG 25 μg/mL for 7 days. Thirteen to thirty flies with three replicates were collected with CO₂ anaesthetization. Activities of superoxide dismutase (SOD) and catalase (Cat) were measured using the SOD Assay Kit-WST (19160, Sigma-Aldrich) and Catalase Activity Colorimetric Assay Kit (BioVision, USA), respectively, as described by manufacturers. Statistical probability was measured using t-test.

SOD activity was determined using the SOD assay kit-WST (Sigma-Aldrich®), which is used to measure the rate of inhibition of the SOD enzyme. The reaction mixtures in the SOD kit were combined with 20 μL of working solution sample and thereafter, were gently shaken before being incubated at 37 °C for 20 min. The inhibition activity of SOD on the reaction of xanthine oxide generated the superoxide with a tetrazolium salt which was then determined by measuring the absorbance of the mixtures at a wavelength of 450 nm using a microplate reader. In this study, the positive control was ascorbic acid (10 mg/mL) in place of the extracts was used.

The SOD activities were measured by an SOD Assay Kit-WST (19160), Sigma. A WST working solution and enzyme working solution were added to blank and study samples in a 96-well plate. After an incubation time of 20 min at 37 °C, the absorbances were read at 450 nm using a plate reader. The SOD activities were calculated and expressed as IU/ml.