Superoxide dismutase (SOD) Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) was used to determine SOD activity in homogenized liver samples. The samples (10 mg, n = 3 per group) were suspended in ultrapure water/PBS and homogenized with frozen metal balls in a TissueLyser ball mill (Qiagen, New York, NY, USA). The homogenate was centrifuged at 1000× g for 20 min. The total protein content in supernatant after homogenization was determined using a BCA Protein Assay Kit according to the manufacturer’s protocol (Sigma-Aldrich, St. Louis, MO, USA). SOD Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) was performed according to the manufacturer’s protocol.
Sod assay kit
Manufactured by Merck Group
Sourced in United States, Germany, Switzerland, Italy, China
The SOD assay kit is a laboratory tool used to measure the activity of the enzyme superoxide dismutase (SOD) in biological samples. It provides a quantitative assessment of SOD levels, which is an important antioxidant enzyme that plays a role in cellular defense against oxidative stress.
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Lab products found in correlation
Catalase assay kit, by Merck Group (6 mentions)
Mda assay kit, by Merck Group (6 mentions)
H2dcfda, by Thermo Fisher Scientific (6 mentions)
Griess reagent kit, by Thermo Fisher Scientific (5 mentions)
Amplex red catalase assay kit, by Thermo Fisher Scientific (4 mentions)
Protease inhibitor, by Cytiva (3 mentions)
Gsh glo glutathione assay kit, by Promega (3 mentions)
Glutathione assay kit, by Merck Group (3 mentions)
Microplate reader, by Thermo Fisher Scientific (3 mentions)
Horac assay kit, by Cell Biolabs (3 mentions)
Tbars assay kit, by Cayman Chemical (3 mentions)
Antioxidant assay kit, by Merck Group (2 mentions)
Senescence cells histochemical staining kit, by Merck Group (2 mentions)
Cellstain double staining kit, by Merck Group (2 mentions)
Lipid peroxidation mda assay kit, by Merck Group (2 mentions)
Catalase, by Merck Group (2 mentions)
Catalase activity assay kit, by Abcam (2 mentions)
Gsh assay kit, by Cayman Chemical (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Trypan blue, by Merck Group (2 mentions)
134 protocols using sod assay kit
1
Superoxide Dismutase Activity Assay
2
Quantifying SOD1 Antioxidant Activity
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SOD1 antioxidant activity was quantified in soluble tissue extracts using a commercial SOD Assay Kit (Cat. no. 19160, Sigma-Aldrich) and potassium cyanide (KCN), as previously described.21 (link) KCN is a potent inhibitor of SOD1 antioxidant activity.22 ,23 (link) Duplicate samples containing 2 µg protein were incubated with 15 mM KCN or 20 mM Tris buffer (pH 7.6; Sigma-Aldrich) for 60 min at room temperature, and SOD activity (U/ml) measured in both samples using the SOD Assay Kit, according to the manufacturer’s instructions. SOD1 activity (U/ml) was calculated by subtracting SOD activity measured in the KCN duplicate (non-SOD1 SOD activity) from the SOD activity measured in the Tris buffer duplicate (total SOD activity). As validation of complete SOD1 inhibition in KCN duplicates, incubation of commercial SOD1 (0–200 U/ml) with 15 mM KCN for 60 min at room temperature completely inhibited SOD1 activity up to ∼50 U/ml (Supplementary Fig. 4 ), whereas the average total SOD activity in age-matched control samples was only 3.91 U/ml in Tris buffer duplicates. The specific activity of SOD1 (U/ml/SOD1 protein) was determined by normalizing SOD1 activity to SOD1 protein levels measured by immunoblotting.
3
Mitochondrial SOD Activity Quantification
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A SOD assay kit from MilliporeSigma (19160-1K-F) was used to determine the SOD activity. Data are expressed as U/mg protein mitochondria.
4
Renal Cortex SOD Activity Assay
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The total superoxide dismutase (SOD) activity was measured in 0.5% homogenates of renal cortex prepared in 0.05 M Tris-HCl buffer (pH 7.4) containing protease inhibitors with the SOD assay kit (MilliporeSigma; 19160-1KT-F), according to the manufacturer’s instructions. The tissue SOD activity was calculated using a SOD standard curve (MilliporeSigma; S7571-15KU) and expressed in units per milligram of proteins (U/mg).
5
Antioxidant Enzyme Activity Assays
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The activity of superoxide dismutase (SOD) was evaluated by the SOD Assay Kit (Sigma-Aldrich, St Louis, MO, USA), following the manufacturer's instructions.
Catalase activity was assayed spectrophotometrically, following the decomposition of H2O2 at 240 nm. The assay mix contained: 50 mM phosphate buffer pH 7.0 and 5 mM H2O2 [22 (link)].
Glutathione peroxidase activity was assayed by the Glutathione Peroxidase Cellular Activity Assay Kit (Sigma-Aldrich, St Louis, MO, USA), following the manufacturer's instructions.
The general antioxidant defenses and the relative level of scavengers were evaluated by the Antioxidant Assay Kit (Sigma-Aldrich, St Louis, MO, USA), following the manufacturer's instructions.
Catalase activity was assayed spectrophotometrically, following the decomposition of H2O2 at 240 nm. The assay mix contained: 50 mM phosphate buffer pH 7.0 and 5 mM H2O2 [22 (link)].
Glutathione peroxidase activity was assayed by the Glutathione Peroxidase Cellular Activity Assay Kit (Sigma-Aldrich, St Louis, MO, USA), following the manufacturer's instructions.
The general antioxidant defenses and the relative level of scavengers were evaluated by the Antioxidant Assay Kit (Sigma-Aldrich, St Louis, MO, USA), following the manufacturer's instructions.
6
Effect of UCB, DHA, and EPA on Antioxidant Enzymes
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Cells were treated with IC50 values of UCB with or without 6-h treatment with DHA (10 μM) or EPA (10 μM). After incubation for 36 h, cells were washed with PBS and treated with trypsin, and the effects of UCB and DHA or EPA on superoxide dismutase (SOD) and catalase (CAT) enzymes activity was detected. SOD and CAT activity was measured using the SOD assay kit (Sigma-Aldrich, USA) and Catalase assay kit (Sigma-Aldrich, USA), respectively.
7
Mitochondrial Dysfunction and Oxidative Stress
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Jag2- or Sirt1-inhibited PASMCs were incubated in six-well plates and treated under normoxic or hypoxic conditions. The mitochondrial membrane potential was assessed with the JC-1 kit (Solarbio Life Sciences, Beijing, China) according to standard procedure. Fluorescent images were acquired using a fluorescence microscope (Olympus, Tokyo, Japan), and mitochondrial membrane potential depolarization was quantified by calculating the percentage of red/green fluorescence using ImageJ software. The mitochondria-targetingsuperoxide anionindicatorMitoSOX (5 mM, Yeasen Biotechnology, Shanghai, China)was used to assay mitochondrial ROS(mtROS) formation.After staining for 15 min, cells were washed with PBS and acell suspensionproduced with trypsin.Finally,mtROS production was detected by flow cytometry and quantified using FlowJo software.To detect oxidative stress in lung tissue, DHE staining was performed according to the manufacturer’s instructions. SOD, MPO, and MDA levels were assessed with a SOD assay kit (Sigma-Aldrich, St. Louis, MO), MPO assay kit (Invitrogen), and MDA assay kit (Sigma-Aldrich) according to the manufacturers’ instructions.
8
Oxidative Stress in Intestinal Tissue
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Intestine tissue homogenate was acquired after 12 or 24 hours of l -arginine exposure as mentioned earlier. The superoxide dismutase (SOD) and malondialdehyde (MDA) levels were detected using the SOD Assay Kit and the Lipid Peroxidation Assay Kit (both from Sigma-Aldrich), respectively, according to the manufacturers’ instructions.
9
Hippocampal SOD Activity Quantification
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The hippocampus of rats was homogenized in PBS with protease inhibitors (Amersham Life Science, Munich, Germany). To remove insoluble material, tissue lysates were sonicated and centrifuged (14,000 rpm, at 4 °C, for 30 min). In the supernatant, total proteins were quantified by the Lowry method [32 (link)]. Volume corresponding to 50 μg of protein was used for total SOD enzymatic activity measurement, by using the SOD assay kit (Sigma–Aldrich) according to manufacturer’s instructions [34 (link)]. Absorbance was measured by using the iMark™ Microplate Absorbance Reader at 450 nm.
10
Senescence and Oxidative Stress Assessment
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Nitric oxide concentration was assessed using reagent kit (Life Technologies) while SOD activity was measured using a SOD Assay kit (Sigma‐Aldrich). ROS were estimated with an H2DCF‐DA (Life Technologies).
The presence of senescence associated β‐galactosidase (β‐gal) in cells was visualized using a commercial kit (Senescence Cells Histochemical Staining Kit; Sigma‐Aldrich) according to the manufacturer's protocol. The percentage of cells expressing β‐gal (stained blue) in regard to β‐gal negative cells was calculated. The number of viable and dead cells was established with Cellstain Double Staining Kit (Sigma‐Aldrich). Viable cells were stained with Calcein‐AM whereas dead cells’ nuclei were with Propidium Iodide.
To assess the mitochondrial membrane's potential, the cell pellets were treated with 1 mmol/L JC‐1 reagent (Life Technologies) in accordance with the manufacturer’ instructions. Obtained results were analysed with CellQuest Pro Software.
The presence of senescence associated β‐galactosidase (β‐gal) in cells was visualized using a commercial kit (Senescence Cells Histochemical Staining Kit; Sigma‐Aldrich) according to the manufacturer's protocol. The percentage of cells expressing β‐gal (stained blue) in regard to β‐gal negative cells was calculated. The number of viable and dead cells was established with Cellstain Double Staining Kit (Sigma‐Aldrich). Viable cells were stained with Calcein‐AM whereas dead cells’ nuclei were with Propidium Iodide.
To assess the mitochondrial membrane's potential, the cell pellets were treated with 1 mmol/L JC‐1 reagent (Life Technologies) in accordance with the manufacturer’ instructions. Obtained results were analysed with CellQuest Pro Software.
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