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Nitroblue Tetrazolium

Nitroblue Tetrazolium (NBT) is a widely used histochemical stain for the detection of superoxide radicals.
It is commonly employed in biochemical and cell biology research to visualize the production of reactive oxygen species, particularly in the context of cellular redox processes and oxidative stress.
NBT is reduced by superoxide to form an insoluble blue formazan precipitate, allowing for the localization and quantification of superoxide generation.
This versatile staining technique has applications in a variety of research areas, including immunology, neuroscience, and plant biology.
Accurate and reproducible NBT staining protocols are crucial for obtaining reliable results and drawing meaningful conclusions from your research.
PubCompare.ai can help optimize your NBT staining protocols by comparing methods from the literature, preprints, and patents, identifying the most effective and reproducible approaches to enhance your research outcomes.

Most cited protocols related to «Nitroblue Tetrazolium»

1
Measuring PMN Oxidative Burst Responses
Several methods are available to measure PMN oxidative burst (8 (link)). Some conventional tests, such as chemiluminescence (9 (link)) and the reduction of cytochrome c (10 (link)), require the isolation of PMN and therefore relatively large amounts of blood. These assays allow monitoring of the production and release of extracellular superoxide anions using photometric or luminometric equipment. More recently, flow cytometry has become widely available to many researchers and therefore assays suitable for this type of equipment have been developed to assess oxidative burst. The assays based on flow cytometry have the advantage that tens of thousands of cells can be assessed in a very short period of time using small volumes of whole blood or isolated PMN (11 (link)). Here, oxidation of specific probes, such as 2′, 7′-dichlorofluorescein diacetate (DCFH) or DHR, to fluorescent derivatives is used to detect superoxide formation in individual cells (Fig. 1).
Besides the methods mentioned above, some additional assays have been described to assess oxidative burst. For example, 3′, 3′-diaminobenzidine (DAB) oxidation and p-nitroblue tetrazolium (NBT) reduction are two simple methods to measure intracellular oxygen radicals or superoxide anions through precipitation reactions (8 (link), 12 (link)). However, these methods are comparatively cumbersome and therefore they are rarely used.
Because they are most reliable in our hands, we have extensively used and optimized the DHR method described above as well as the more traditional SOD-inhibitable reduction of cytochrome c to characterize oxidative burst activity in PMN (7 , 13 (link)).
Chen Y, & Junger W.G. (2012). Measurement of Oxidative Burst in Neutrophils. Methods in molecular biology (Clifton, N.J.), 844, 115-124.
Publication 2012
Biological Assay BLOOD Blood Volume Cells Chemiluminescence Cytochromes c derivatives Flow Cytometry Nitroblue Tetrazolium Photometry Protoplasm Reactive Oxygen Species Respiratory Burst Superoxides Tetranitrate, Pentaerythritol
2
Two-Color in Situ Hybridization for Mouse Brain
The first-pass single-color ISH analysis was performed essentially as described previously29 (link). The accession numbers of the DNA templates used for probe generation and the genomic sequence (mm9 assembly) that was amplified by PCR for template generation are listed in Supplementary Table 3. For the two-color section ISH analysis, maternal animals were anesthetized and embryos were immediately dissected out. The brains were removed, fixed overnight in 30% sucrose/4% paraformaldehyde (vol/vol) and sectioned in the coronal plane on a Leica sledge microtome at 40 μm. Sections were individually mounted on slides and processed for ISH to visualize expression of various genes. Single- or two-color nonradioactive ISH was performed using previously described method with some modifications32 (link). Glass slides with sections were fixed by 4% paraformaldehyde and treated with proteinase K (Roche). Sections were then fixed again and hybridized with digoxigenin-labeled or fluorescein-labeled probes (Roche) at 70 °C overnight. Excess probes were washed out and sections were blocked with lamb serum and incubated with solution containing alkaline phosphatase–conjugated with digoxigenin-labeled or fluorescein-labeled antibodies (Roche). Color was developed with combinations of the chromagens nitroblue tetrazolium (Nacalai, 350 mg ml−1) and 5-bromo, 4-chloro, 3-indolylphosphate for blue staining or tetranitroblue tetrazolium (Research Organics, 350 mg ml−1) and 5-bromo, 4-chloro, 3-indolylphosphate for brown staining. The probes used for this analysis that gave good signal using two-color ISH are listed in Supplementary Table 4, whereas probes that were tested, but did not give good signal, are included in Supplementary Table 3. All two-color ISH data using candidate genes and Shh can be accessed at http://blackshaw.bs.jhmi.edu and in the Mouse Gene Expression Database at Jackson Labs (http://www.informatics.jax.org/expression.shtml).
Shimogori T., Lee D.A., Miranda-Angulo A., Yang Y., Wang H., Jiang L., Yoshida A.C., Kataoka A., Mashiko H., Avetisyan M., Qi L., Qian J, & Blackshaw S. (2010). A genomic atlas of mouse hypothalamic development. Nature neuroscience, 13(6), 767-775.
Publication 2010
Alkaline Phosphatase Animals Antibodies blue 4 Brain Digoxigenin Embryo Endopeptidase K Fluorescein Gene Expression Genes Genome Mice, Laboratory Microtomy Mothers Nitroblue Tetrazolium paraform Serum Sheep Sucrose Tetrazolium Salts
3
Quantitative In Situ Detection of Superoxide
The nitroblue tetrazolium (NBT) (N6876, Sigma-Aldrich) staining method of Rao and Davis [74 (link)] was modified as follows for in situ detection of superoxide radical. Three week-old plantlets were transferred for 12, 24, 48 or 72 hours to the different control and treatment media described above (M, S, MA and SA). Plantlets, prior to the transfer and at the end of the treatment, were immersed and infiltrated under vacuum with 3.5 mg ml-1 NBT staining solution in potassium phosphate buffer (10 mM) containing 10 mM NaN3. After infiltration, stained plantlets were bleached in acetic acid-glycerol-ethanol (1/1/3) (v/v/v) solution at 100°C during 5 min. Plantlets were then stored in a glycerol-ethanol (1/4) (v/v) solution until photographs were taken. O2.- was visualized as a blue color produced by NBT precipitation. A modified version of previously described assays for superoxide quantification was used [75 (link),76 (link)]. Briefly, NBT-stained plantlets were ground in liquid nitrogen, the formazan content of the obtained powder was solubilized in 2 M KOH-DMSO (1/1.16) (v/v), and then centrifuged for 10 min at 12,000 g. The A630 was immediately measured, and compared with a standard curve obtained from known amounts of NBT in the KOH-DMSO mix. Experiments were repeated four times on at least 15 plantlets.
Ramel F., Sulmon C., Bogard M., Couée I, & Gouesbet G. (2009). Differential patterns of reactive oxygen species and antioxidative mechanisms during atrazine injury and sucrose-induced tolerance in Arabidopsis thaliana plantlets. BMC Plant Biology, 9, 28.
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Publication 2009
Acetic Acid Biological Assay Buffers Ethanol Formazans Glycerin Nitroblue Tetrazolium Nitrogen potassium phosphate Powder Sodium Azide Sulfoxide, Dimethyl Superoxides Vacuum
4
Enzyme Activity Assays for Ginsenoside Biosynthesis
Amylase (AMY, EC 3.1.1.2) activity was detected by the 3,5-dinitrosalicylic acid colorimetric method (Hao et al., 2007 ). Malate dehydrogenase (MDH, EC 1.1.1.37) activity was examined as described by Husted and Schjoerring (1995 (link)), with some modifications. Ten microliter samples were added to a 3 ml reaction mixture containing 0.17 mM oxalacetic acid and 0.094 mM β-NADH disodium salt in 0.1 M Tris buffer, pH 7.5. The reaction was measured by the decrease in absorbance at 340 nm for 180s in a spectrophotometer (Hitachi U-2001 Japan), the same reaction system only with sample buffer added in was used as a blank. Superoxide dismutase (SOD, EC 1.14.1.1) activity was measured according to the method of Zhang and Kirkham (1996 (link)), and Xu and Huang (2004 (link)). One unit of SOD activity is defined as the amount of SOD required to cause 50% inhibition of nitroblue tetrazolium (NBT) reduction at 560 nm min-1. Catalase (CAT, EC 1.11.1.6) and peroxidase (POD, EC.1.11.1.7) activity were determined based on the method of Chance and Maehly (1955 (link)) as described in detail for creeping bentgrass in Xu and Huang (2004 (link)). Enzyme activities were based on the absorbance change of the reaction solution per minute at a given wavelength for each enzyme: CAT at 240 nm and POD at 470 nm.
The activities of farnesyl diphosphate synthase (FDPS, EC. 2.5.1.10), cycloartenol synthase (CAS, EC. 5.4.99.8), squalene epoxidases (SE, EC:1.14.13.132), and squalene synthase (SS, EC. 2.5.1.21) involved in ginsenosides biosynthesis, were quantified by an indirect competitive enzyme-linked immunosorbent assay (ELISA). The optical density (OD) values of each sample were read by a BioTek ELx800 microplate reader at 450 nm. The primary concentration of each test sample was calculated from the linear regression equation based on the OD values of the standards.
Ma R., Sun L., Chen X., Mei B., Chang G., Wang M, & Zhao D. (2016). Proteomic Analyses Provide Novel Insights into Plant Growth and Ginsenoside Biosynthesis in Forest Cultivated Panax ginseng (F. Ginseng). Frontiers in Plant Science, 7, 1.
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Publication 2016
Acids Agrostis Amylase Anabolism Buffers Catalase Colorimetry cycloartenol synthase Enzyme-Linked Immunosorbent Assay enzyme activity Enzymes Farnesyltransferase, Farnesyl-Diphosphate Geranyltranstransferase Ginsenosides NADH Nitroblue Tetrazolium Oxaloacetic Acid Peroxidase Psychological Inhibition Sodium Chloride Squalene Monooxygenase Superoxide Dismutase Tromethamine
5
Antioxidant Enzyme Activity Assays
Fresh plant material (1g) was homogenized in 100 mM Tris-HCl (pH 7.5) in presence of DTT (Dithiothreitol, 5 mM), MgCl2 10 mM, Ethylenediaminetetraacetic acid (EDTA, 1 mM), magnesium acetate 5 mM, Polyvinylpyrolidone (PVP-40 1.5%), phenylmethanesulfonyl fluoride (PMSF 1 mM) and aproptinin 1 μgmL-1. After the filtration, the homogenate was centrifuged at 10,000 rpm for 15 min. The supernatant collected after centrifugation served as enzyme source. For the analysis of APX activity, tissues were separately homogenized with 2 mM AsA. All experiments were performed at 4°C.
Activity of SOD was estimated according to Kono (1978) (link) following the photo reduction of nitroblue tetrazolium (NBT). The absorbance was recorded spectrophotometerically (Beckman 640 D, USA) at 540 nm. SOD unit is the quantity of enzyme that hamper 50% photoreduction of NBT and is expressed as EU mg-1 protein.
The activity of POD was estimated according to the method proposed by Putter and Becker (1974) . The rate of production of oxidized guaiacol was estimated spectrophotometerically (Beckman 640 D, USA) at 436 nm. The activity of POD was expressed as EU mg-1 protein.
Catalase activity was estimated by the method of Aebi (1984) (link). The OD was taken spectrophotometerically (Beckman 640 D, USA) at 240 nm and the activity was expressed as EU mg-1 protein.
For the determination of APX activity, the procedure of Nakano and Asada (1981) was used. The OD was recorded at 265 nm by spectrophotometer (Beckman 640 D, USA) and the activity was expressed as EU mg-l protein.
Sirhindi G., Mir M.A., Abd-Allah E.F., Ahmad P, & Gucel S. (2016). Jasmonic Acid Modulates the Physio-Biochemical Attributes, Antioxidant Enzyme Activity, and Gene Expression in Glycine max under Nickel Toxicity. Frontiers in Plant Science, 7, 591.
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Publication 2016
Catalase Centrifugation Dithiothreitol Edetic Acid Enzymes Filtration Guaiacol magnesium acetate Magnesium Chloride Nitroblue Tetrazolium Phenylmethylsulfonyl Fluoride Plants Proteins PVP 40 Tissues Tromethamine

Most recents protocols related to «Nitroblue Tetrazolium»

1
Quantifying Oxidative Stress Markers in Plasma
In plasma samples, the following oxidative stress markers were measured: nitrite (NO2), superoxide anion radical (O2−), hydrogen peroxide (H2O2), and the index of lipid peroxidation (measured as TBARS – thiobarbituric acid reactive substances).
Nitric oxide decomposes rapidly to form stable metabolite nitrite/nitrate products. The nitrite level was measured and used as an index of nitric oxide (NO) production using the Griess reagent. A total of 0.5 ml of plasma was precipitated with 200 μl of 30% sulphosalicylic acid, vortexed for 30 min, and centrifuged at 3000 × g. Equal volumes of supernatant and Griess reagent containing 1% sulphanilamide in 5% phosphoric acid/0.1% naphthalene ethylenediamine dihydrochloride were added and incubated for 10 min in the dark, and the sample was measured at 543 nm. The nitrite levels were calculated using sodium nitrite as the standard [13 (link)].
The O2− concentration was measured after the reaction of nitro blue tetrazolium in Tris buffer with the plasma at 530 nm. Distilled water served as the blank [14 ].
The measurement of H2O2 is based on the oxidation of phenol red by H2O2 in a reaction catalysed by horseradish peroxidase (HRPO). Two hundred μl of plasma was precipitated with 800 ml of freshly prepared phenol red solution, followed by the addition of 10 μl of (1:20) HRPO (made ex tempore). Distilled water was used as the blank instead of the plasma sample. H2O2 was measured at 610 nm [15 (link)].
The degree of lipid peroxidation in the plasma samples was estimated by measuring TBARS using 1% thiobarbituric acid in 0.05 NaOH, incubated with the plasma at 100 °C for 15 min, and measured at 530 nm. Distilled water served as the blank [16 (link)].
The activity of the following antioxidants in the lysate was determined: reduced glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD). The level of reduced glutathione was determined based on GSH oxidation with 5,5-dithiobis-6,2-nitrobenzoic acid using a method by Beutler [17 ]. The CAT activity was determined according to Aebi [18 (link)]. The lysates were diluted with distilled water (1:7 v/v) and treated with chloroform-ethanol (0.6:1 v/v) to remove haemoglobin, and then 50 μl of CAT buffer, 100 μl of sample and 1 ml of 10 mM H2O2 were added to the samples. The detection was performed at 360 nm. SOD activity was determined by the epinephrine method of Beutler [19 (link)]. Lysate (100 μl) and 1 ml carbonate buffer were mixed, and then 100 μl of epinephrine was added. The detection was performed at 470 nm.
Radoman K., Zivkovic V., Zdravkovic N., Chichkova N.V., Bolevich S, & Jakovljevic V. (2023). Effects of dandelion root on rat heart function and oxidative status. BMC Complementary Medicine and Therapies, 23, 78.
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Publication 2023
Anions Antioxidant Activity Buffers Carbonates Catalase Chloroform Epinephrine Ethanol ethylenediamine dihydrochloride Griess reagent Hemoglobin Horseradish Peroxidase Lipid Peroxidation naphthalene Nitrates Nitrites Nitrobenzoic Acids Nitroblue Tetrazolium Oxidative Stress Oxide, Nitric Peroxide, Hydrogen Phosphoric Acids Plasma Reduced Glutathione Sodium Nitrite Sulfanilamide sulfosalicylic acid Superoxide Dismutase Superoxides thiobarbituric acid Thiobarbituric Acid Reactive Substances Tromethamine
2
Leaf Nutrient and Antioxidant Analysis
For each treatment, 30 healthy and mature leaves were selected, washed, and dried. Following a 30-min incubation at 105 °C, the leaves were dried at 75 °C, crushed, and screened. The ground leaves were collected in a self-sealing bag and stored in a dryer. Leaf samples were boiled in H2SO4–HClO4 and HNO3–HClO4 solutions and then the nutrient element contents were determined using AutoAnalyzer 3 with XY-2 Sampler (SEAL Analytical, UK) and an inductively coupled plasma emission spectrometer (Prodigy Spec, Teledyne, USA).
On August 10, 10 randomly selected leaves (per treatment) were obtained from OY saplings to measure the catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) contents as previously described (Wang et al., 2018 (link)). CAT was determined by monitoring the decomposition of H2O2. SOD was assayed by monitoring the inhibition of the photochemical reduction of nitro blue tetrazolium. POD was determined by monitoring the oxidation reaction of guaiacol.
Li G., Li J., Zhang H., Li J., Jia L., Zhou S., Wang Y., Sun J., Tan M, & Shao J. (2023). ASSVd infection inhibits the vegetative growth of apple trees by affecting leaf metabolism. Frontiers in Plant Science, 14, 1137630.
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Publication 2023
Catalase Guaiacol Nitroblue Tetrazolium Nutrients Peroxidase Peroxide, Hydrogen Phocidae Plasma Prodigy Psychological Inhibition Superoxide Dismutase
3
Superoxide Radical Scavenging Assay

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Publication 2023
Biological Assay Coenzyme I Formazans Methylphenazonium Methosulfate NADH Nitroblue Tetrazolium Psychological Inhibition Rutin Superoxides
4
Quantifying Intracellular ROS Levels
To investigate the dominant mechanism of the proposed treatment, both the control and treated US were placed in a 15 mL conical tube with 1 mL distilled water, sonicated for 20 min, and vortexed for 5 min to detach the bacteria in the US. A 1 mg solution of nitro blue tetrazolium (NBT) was added to the tube and incubated for 30 min in the dark. After 0.1 M HCl was added to the solution to inhibit bacterial interaction with NBT, all tubes were centrifuged at 12,000 × g for 5 min. To release intercellular ROS, the pellets were first treated with 800 mL of saline and 400 mL of dimethyl sulfoxide (DMSO). To estimate ROS generation, 200 μL of each sample was placed in a 96-well plate and measured at 575 nm (N = 5 per condition). The collected ROS generation was normalized by the absorbance of control samples to exclude any experimental errors caused by the sonication procedure. The following equation was used to determine the level of intercellular ROS amplification (Ri): Ri=At-AcAc×100 where Ac is the absorbance of the control and At is the absorbance of the treated sample.
Maknuna L., Tran V.N., Lee B.I, & Kang H.W. (2023). Inhibitory effect of 405 nm laser light on bacterial biofilm in urethral stent. Scientific Reports, 13, 3908.
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Publication 2023
Bacteria Cardiac Arrest Nitroblue Tetrazolium Pellets, Drug Saline Solution Sulfoxide, Dimethyl
5
Assessing Wheat Seedling Stress Response
Evans blue staining was employed to determine cell death in wheat seedling leaves [38 (link)]. The in situ generation of superoxide radical (O2−.) and hydrogen peroxide (H2O2) in wheat seedling leaves were evaluated by histochemical staining with nitroblue tetrazolium (NBT) and 3, 3’-diaminobenzidine (DAB) staining, respectively [38 (link)]. Determination of the the activities of SOD, POD, and CAT and a TUNEL assay were carried out as described by Yue et al. (2021) [38 (link)]. The activity of caspase-3 was determined according to Yue et al. (2022) [47 ].
Yue J.Y., Jiao J.L., Wang W.W., Jie X.R, & Wang H.Z. (2023). Silencing of the calcium-dependent protein kinase TaCDPK27 improves wheat resistance to powdery mildew. BMC Plant Biology, 23, 134.
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Publication 2023
Biological Assay Caspase 3 Cell Death Evans Blue In Situ Nick-End Labeling Nitroblue Tetrazolium Peroxide, Hydrogen Superoxides Triticum aestivum

Top products related to «Nitroblue Tetrazolium»

1
Nitroblue tetrazolium by Merck Group
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Nitroblue tetrazolium is a chemical compound used in various laboratory applications. It serves as an indicator for the detection of reducing substances, particularly enzymes that catalyze redox reactions. The compound undergoes reduction to form a dark blue, insoluble formazan product, which can be quantified to measure the activity or presence of the target analyte.
2
Nitroblue tetrazolium nbt by Merck Group
Sourced in United States, Germany
Nitroblue tetrazolium (NBT) is a water-soluble tetrazolium salt used in biochemical and cellular assays. It serves as an electron acceptor, and its reduction to a blue-colored formazan product can be used to detect the presence of enzymes or reactive oxygen species in biological samples.
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Bovine serum albumin (bsa) by Merck Group
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Bovine serum albumin (BSA) is a common laboratory reagent derived from bovine blood plasma. It is a protein that serves as a stabilizer and blocking agent in various biochemical and immunological applications. BSA is widely used to maintain the activity and solubility of enzymes, proteins, and other biomolecules in experimental settings.
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Gallic acid by Merck Group
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Gallic acid is a naturally occurring organic compound that can be used as a laboratory reagent. It is a white to light tan crystalline solid with the chemical formula C6H2(OH)3COOH. Gallic acid is commonly used in various analytical and research applications.
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Ascorbic acid by Merck Group
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Ascorbic acid is a chemical compound commonly known as Vitamin C. It is a water-soluble vitamin that plays a role in various physiological processes. As a laboratory product, ascorbic acid is used as a reducing agent, antioxidant, and pH regulator in various applications.
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Trichloroacetic acid is a colorless, crystalline chemical compound used in various laboratory applications. It serves as a reagent and is commonly employed in analytical chemistry and biochemistry procedures. The compound's primary function is to precipitate proteins, making it a useful tool for sample preparation and analysis.
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Riboflavin by Merck Group
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Riboflavin, also known as vitamin B2, is a water-soluble vitamin that is commonly used in laboratory settings. It serves as a core component in various biological processes, including energy metabolism and cellular respiration. Riboflavin plays a crucial role as a cofactor for enzymes involved in the conversion of food into energy. This product is often used in research and analytical applications where its specific properties and functions are required.
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2 2 diphenyl 1 picrylhydrazyl (dpph) by Merck Group
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DPPH is a chemical compound used as a free radical scavenger in various analytical techniques. It is commonly used to assess the antioxidant activity of substances. The core function of DPPH is to serve as a stable free radical that can be reduced, resulting in a color change that can be measured spectrophotometrically.
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Nbt bcip by Roche
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NBT/BCIP is a chromogenic substrate used for the detection and visualization of alkaline phosphatase activity in various biological and biochemical assays. The product consists of two components, nitro-blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt (BCIP), which together produce a dark-purple insoluble precipitate upon enzymatic cleavage by alkaline phosphatase.
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Bcip nbt by Merck Group
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BCIP/NBT is a chromogenic substrate used for the detection and visualization of alkaline phosphatase activity in various biological applications, such as immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assay (ELISA).

More about "Nitroblue Tetrazolium"

Nitroblue tetrazolium (NBT) is a widely used histochemical stain for detecting superoxide radicals, a reactive oxygen species (ROS).
This versatile staining technique is commonly employed in biochemical and cell biology research to visualize and quantify the production of ROS, particularly in the context of cellular redox processes and oxidative stress.
When superoxide is present, NBT is reduced to form an insoluble blue formazan precipitate, allowing for the localization and quantification of superoxide generation.
NBT staining has applications in a variety of research areas, including immunology, neuroscience, and plant biology.
Accurate and reproducible NBT staining protocols are crucial for obtaining reliable results and drawing meaningful conclusions.
Factors such as bovine serum albumin, gallic acid, ascorbic acid, trichloroacetic acid, riboflavin, and DPPH can affect the NBT staining process and should be considered when optimizing protocols.
PubCompare.ai can help enhance your NBT research by providing AI-driven protocol optimization.
This powerful tool allows you to easily locate and compare NBT staining methods from the literature, preprints, and patents, identifying the most effective and reproducible approaches to improve your research outcomes.
Whether you're working with NBT/BCIP or BCIP/NBT, PubCompare.ai can help you fine-tune your protocols and maximize the reliability and accuracy of your Nitroblue tetrazolium-based studies.