Superoxide dismutase assay kit

Peripheral venous blood (2 mL) was obtained from each participant into a sterile tube containing heparin (an anticoagulant). The blood was centrifuged at 3500 revolutions/min for 10 min; then, the supernatant was transferred to a fresh tube and stored at −20°C until analysis. Each sampling tube was given a unique label, stored in a portable refrigerator, and transferred to the laboratory for processing within 3 h of collection. The investigators did not know which participant was allocated to which group. The superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-px) activity, and malondialdehyde (MDA) concentration in each plasma sample were determined to allow oxidative stress to be assessed. The SOD activity, GSH-px activity, and MDA concentration were prepared using a superoxide dismutase assay kit (Beyotime Biotechnology, Shanghai, China), a lipid oxidation assay kit (Beyotime Biotechnology), and a glutathione peroxidase assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), respectively, and the prepared samples were analyzed using a DR5000 ultraviolet/visible spectrophotometer.

Fluorescent-labeled anti-mouse mAbs APC-CD11c (N418), PE-CD40 (1C10), PE-CD80 (16-10A1), FITC-CD86 (GL1), FITC-MHCII (M5/114.15.2), PE-CCR7 (4B12), FITC-CD4 (GK1.5), APC-IL-4 (11B11), PE-IFN-γ (XMG1.2), or respective isotype controls were purchased from eBioscience (San Diego, CA, USA). Mouse CD36 blocking antibody [JC63.1] and control IgA antibody were from Abcam (Cambridge, USA). Rabbit anti-mouse pERK, ERK, pJNK, JNK, pP38, P38, IκBα, p65, LaminB1, GAPDH, goat anti-rabbit IgG-DY488 and goat anti-rabbit IgG-HRP were from Bioworld (St. Louis Park, MN, USA). RPMI 1640 medium, DAPI, FITC-OVA, and CFSE were from Invitrogen (Grand Island, NY, USA). Fetal bovine serum (FBS) was from Hyclone (Thermo, Melbourne, Australia). Recombinant GM-CSF, IL-4, IL-2, and CCL19 were from Peprotech (Rocky Hill, NJ). LPS (from Escherichia coli 026: B6), 2′,7′-dichlorodi hydro fluorescein diacetate (DCFDA) and β-carotene were from Sigma-Aldrich (St Louis, MO, USA). PD 98, 059, SP600125, BAY 11-7082, Bradford assay kit, Nitric Oxide assay kit, Superoxide dismutase assay kit, and Nuclear and Cytoplasmic Protein Extraction Kit were from Beyotime (Jiangsu, China). Mouse IL-6, IL-10, IL-12p70, TNFα ELISA Kit and Cell Counting Kit-8 (CCK8) were from Boster (Wuhan, China).

We used superoxide dismutase assay kit (Beyotime, S0109) to measure SOD activity, used lipid peroxidation MDA assay kit to measure MDA (Beyotime, S0131S), used tissue iron content assay kit to measure Fe²⁺ and used micro reduced glutathione assay kit (Solarbio, BC1175) to measure GSH. All the procedures were conducted according to the manufacturer's instructions.

For these assays, IPEC-J2 cell number was optimized at 1 × 10⁶ per well in 6-well plate. Cells were washed twice with cold PBS and harvested in 5% metaphosphoric acid using a cell scraper. Harvested cells were lysed by sonication on ice, and the supernatant of the centrifugate (1300 ×g at 4°C for 15 min) was collected. GSH levels were measured colorimetrically using a GSH assay kit (Nanjing Jiancheng Institute of Bioengineering) according to the manufacturer's protocol and normalized to the protein concentration in the lysate. This assay is based on the spectrophotometric measurement of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), the product of a reaction with GSH. The yellow derivative 5′-thio-2-nitrobenzoic acid (TNB) was measured by detecting absorbance at 412 nm using a microplate reader. SOD1 activity was determined by measuring inhibition of the reduction of the water-soluble tetrazolium salt, WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt), which produces a water-soluble formazan dye upon reduction with O₂^−•. Determinations were performed with a Superoxide Dismutase Assay Kit (Beyotime Biotechnology, ShangHai, China) specific to SOD1.