Fungal cells were grown in GMM(Pro) medium, and the stress-treated cell extracts were obtained using the same method described above. After quantifying the amounts of the proteins, the proteins were removed from the cell extracts using Amicon Ultra 0.5-mL filters (Merck, Darmstadt, Germany). The resultant samples were transferred to a 96-well plate, and the NO level was quantified using an NO2/NO3 Assay Kit-C II(Colorimetric) ~ Griess Reagent Kit ~ (Dojindo) at 540 nm.
No2 no3 assay kit c 2
Manufactured by Dojindo Laboratories
Sourced in Japan
The NO2/NO3 Assay Kit-C II is a laboratory equipment product from Dojindo Laboratories. It is designed for the determination of nitrite (NO2-) and nitrate (NO3-) concentrations in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Amicon ultra 0.5 ml filter, by Merck Group (1 mentions)
Griess reagent kit, by Dojindo Laboratories (1 mentions)
Automatic microplate reader, by Bio-Rad (1 mentions)
Creatinine urinary colorimetric assay kit, by Cayman Chemical (1 mentions)
Il 1β, by R&D Systems (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
Il 10, by R&D Systems (1 mentions)
5 protocols using no2 no3 assay kit c 2
1
Nitric Oxide Quantification in Fungal Cells
2
Ar-CAP Irradiation and NO2/NO3 Assay
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Briefly, 1 ml milli-Q water was added to 24-well tissue culture plate, and Ar-CAP irradiation was performed for 1 min. After treatment, 80 μL sample was taken out to 96-well plate, and a NO2/NO3 assay was performed using a NO2/NO3 Assay Kit-CII (Dojindo, Kumamoto, Japan), the optical density was measured at 570 nm by an automatic microplate reader (Bio-Rad, Hercules, CA).
3
Biomarker Evaluation in Plasma and Urine
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Plasma and urine were used for measurement of NO2/NO3, 8-hydroxy-2′-deoxyguanosine (8-OHdG), hexanoyl-lysine (HEL), and myeloperoxidase (MPO) by corresponding commercial kit [NO: NO2/NO3 Assay Kit-C II (DOJINDO LABORATORIES, Kumamoto, Japan); 8-OHdG: New 8-OHdG Check ELISA (Japan Institute for the Control of Aging, NIKKEN SEIL Co., Ltd. (JaICA) Shizuoka, Japan); HEL: HEL ELISA kits (JaICA); and MPO: Human serum MPO and urine MPO ELISA kits (JaICA)]. Urinary NO2/NO3 level was corrected by creatinine equivalent, which was measured by using Creatinine (urinary) Colorimetric Assay Kit (Cayman CHEMICAL, Ann Arbor, MI, USA).
Analyses of hematologic biochemical markers including creatinine, total protein, blood urea nitrogen, glucose, lactate dehydrogenase, alkaline phosphatase, γ- glutamyltranspeptidase, aspartate aminotransferase, alanine aminotransferase, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, Na+, Cl−, K+, white blood cells, red blood cells, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean hemoglobin concentration, and platelet were measured by LSI Medience Co. (Tokyo, Japan).
Analyses of hematologic biochemical markers including creatinine, total protein, blood urea nitrogen, glucose, lactate dehydrogenase, alkaline phosphatase, γ- glutamyltranspeptidase, aspartate aminotransferase, alanine aminotransferase, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, Na+, Cl−, K+, white blood cells, red blood cells, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean hemoglobin concentration, and platelet were measured by LSI Medience Co. (Tokyo, Japan).
4
Nitric Oxide Levels in Epilepsy Mice
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Mutant epilepsy-prone EL mice manifesting no seizures before 5 weeks of age were used, with ddY mice serving as controls (The El mouse was established in Japan as a genetically predisposed epilepsy model from the hydrocephalic mutant of the ddY strain 9 ). The brain redox was studied.
Five EL mice and 5 control ddY mice were sacrificed by decapitation, and the brains were removed and placed on ice. The parietal cortex and the hippocampus were excised and weighed (8-20 mg). To obtain brain tissue homogenates, 20 mM Tris-HCl (pH 8.0) was added.
An NO2/NO3 Assay Kit-CII (Dojindo, Kumamoto, Japan) was used to determine NO levels. Briefly, the Griess reaction was used to determine NO2 levels spectrophotometrically at 540 nm 10 . For NO3 reduction, samples were incubated in the presence of nitrate reductase, NADPH and FAD. Since all NO fractions in our samples were converted to NO2, their levels were determined spectrophotometrically to serve as total NO (NO2+NO3) levels. The Bradford assay 11 was used to measure total protein concentrations in each sample with a Bio-Rad reagent (Bio-Rad, Richmond, California, United States).
Data are presented as mean ± standard error of the mean, and two way ANOVA was used to determine the statistical significance of differences in each parameter in mice of the same age; a level of p < 0.01 was considered significant.
Five EL mice and 5 control ddY mice were sacrificed by decapitation, and the brains were removed and placed on ice. The parietal cortex and the hippocampus were excised and weighed (8-20 mg). To obtain brain tissue homogenates, 20 mM Tris-HCl (pH 8.0) was added.
An NO2/NO3 Assay Kit-CII (Dojindo, Kumamoto, Japan) was used to determine NO levels. Briefly, the Griess reaction was used to determine NO2 levels spectrophotometrically at 540 nm 10 . For NO3 reduction, samples were incubated in the presence of nitrate reductase, NADPH and FAD. Since all NO fractions in our samples were converted to NO2, their levels were determined spectrophotometrically to serve as total NO (NO2+NO3) levels. The Bradford assay 11 was used to measure total protein concentrations in each sample with a Bio-Rad reagent (Bio-Rad, Richmond, California, United States).
Data are presented as mean ± standard error of the mean, and two way ANOVA was used to determine the statistical significance of differences in each parameter in mice of the same age; a level of p < 0.01 was considered significant.
5
Cytokine Profiling in Splenocyte Cultures
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The concentrations of the LPS‐induced cytokines, NO, IFN‐γ, IL‐1β, IL‐6 and IL‐10 in the culture medium of splenocytes were measured after 4, 8, 16, and 24 h. NO is hydrolyzed in medium, and this is followed by the formation of NO stable metabolites (nitrate + nitrite [NOx]). NOx concentrations were measured using the Griess method (NO2/NO3 Assay Kit‐CII; Dojindo, Kumamoto, Japan). Enzyme‐linked immunosorbent assay was carried out to measure the levels of TNF‐α (IBL, Gumma, Japan), IFN‐γ (R&D systems, Minneapolis, MN, USA), IL‐1β (R&D systems), IL‐6 (Arigobio, Hsinchu, Taiwan) and IL‐10 (R&D systems).
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