Sod colorimetric activity kit

RAW 264.7 cells were treated with OS (62.5 μg/mL, 125 μg/mL, and 250 μg/mL) and incubated for 48 h. The supernatant was collected by centrifugation (15,000 rpm, 10 min, 4 °C). SOD activity was evaluated according to the SOD colorimetric activity kit protocol (EIASODC, Invitrogen Co., Carlsbad, CA, USA). In brief, each 10 μL of supernatant was added to 50 μL of the substrate solution with 25 μL of a xanthine oxidase solution in a new plate and the plate was kept at RT for 20 min. The absorbance was measured at 450 nm using a microplate reader (Molecular Devices). The SOD activity was calculated by the standard curve.

Superoxide Dismutase (SOD) activity of worm lysates was measured using the SOD Colorimetric Activity Kit (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. The assay measures all types of SOD activity, including Cu/Zn, Mn, and FeSOD types, through the effect of the xanthine oxidase, which generates superoxide in the presence of oxygen. The colored product was read at 450 nm with a SPECTROstar nano (BMG Labtech, Ortenberg, Germany). The values of SOD were calculated from the standard curve, and data were corrected for mg of protein of each sample.

To assess antioxidative enzyme activities, 10-day-old worms were ground in liquid nitrogen. Superoxide dismutase (SOD) and catalase activity levels were measured with the SOD colorimetric activity kit (Invitrogen, CA, USA) or Catalase activity colorimetric/fluorometric assay kit (Biovision, CA, USA), respectively. Worm pellet samples were assessed according to manufacturer’s protocol in triplicate. SOD and catalase activity were determined using standard curves followed by normalization to protein concentration using the BCA protein assay (Pierce, IL, USA).

Enzymatic colorimetric kits were used for the general determination of plasma total cholesterol (TC), triglycerides (TG), glucose (QCA, Barcelona, Spain) and non-esterified free fatty acids (NEFAs, WAKO, Neuss, Germany).
To evaluate oxidative stress, we measured the markers of lipid oxidative damage by measuring malondialdehyde (MDA, TBARS assay kit, Cayman Chemical Company, Ann Arbor, MI, USA) and 8-isoprostane (8-isoprostane ELISA kit, Cayman Chemical Company, Ann Arbor, MI, USA). To know the antioxidant capacity of the subjects, we quantified the activity of the main antioxidant enzyme, superoxide dismutase (SOD, SOD Colorimetric Activity Kit, Thermo Fisher Scientific, Waltham, MA, USA). The overall inflammatory response was measured with the level of the monocyte chemoattractant protein-1 (MCP-1, MCP-1 Rat Instant ELISA™ Kit, Thermo Fisher Scientific, Waltham, MA, USA) one of the main pro-inflammatory cytokines. The manufacturer’s protocol was followed for all the determinations.

Superoxide dismutase (SOD) activity was measured using a SOD colorimetric activity kit and the manufacturer’s protocol (Thermo Fisher Scientific). This assay measures all types of SOD activity, including Cu/Zn, Mn, and FeSOD types. Samples (plastids/mitochondria/protoplasts) were diluted in colored sample diluent and added to the wells of a 96-well plate. The substrate was added followed by Xanthine Oxidase Reagent and incubation at room temperature for 20 min. Superoxide is generated by the xanthine oxidase that converts a colorless substrate to a yellow-colored product, which was quantified at 450 nm by an absorbance assay. A SOD standard curve was used for all samples.

On day 21, the UB tissues were homogenized with 100 μL ice-cold tissue RIPA lysis buffer. The homogenate was incubated at 4 °C for 30 min and centrifuged at 12,000× g at 4 °C for 20 min. The supernatants were obtained as the total protein extracts. Total protein concentration was measured with the bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific, Waltham, MA, USA).
TNF-α, IL-6, IL-8, IL-1β, and NF-κB levels were measured in UB tissues homogenates using ELISA kits for rats (Biolegend, San Diego, CA, USA). The supernatants nuclear extracts were prepared using the NE-PER Nuclear and Cytoplasmic Extraction system (Thermo Fisher Scientific, Waltham, MA, USA). For each assay, 10 μg of nuclear extracts from UB tissues’ homogenates were used in a TransAM NF-κB p65 ELISA kit (Active Motif, Carlsbad, CA, USA). The level of malondialdehyde (MDA) was determined using the Lipid Peroxidation MDA assay kit (Colorimetric/Fluorometric) (Cat# ab118970, Abcam, Cambridge, MA, USA). Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were determined using the SOD Colorimetric activity kit (Cat# EIASODC, Thermo Fisher Scientific, Waltham, MA, USA) and GSH-Px ELISA kit (Cat#11352, Cusabio Biotech Co., Ltd., Wuhan, China), respectively. All steps were completed according to the manufacturer’s instructions.