This assay is widely used to measure the superoxide release of cells. AM cells isolated from lavage fluid were cultured in 96-well plates and incubated at 37°C with 5% CO2 overnight. One microgram/milliliter nitroblue tetrazolium (NBT) dye (Sigma, St Louis, MO) was added to each well of the plate following the manufacturer's instructions. The yellow color NBT can change to blue upon reduction by released superoxide (38 (link)). The reaction was terminated by adding 10 μl of stop solution as above. The plate was left at room temperature overnight for complete dissolution of dye product, and a multiscan plate reader to quantify the dye conversion read the absorbance at 560 nm. Each experiment was conducted in triplicate (37 (link)).
Nitroblue tetrazolium nbt dye
Manufactured by Merck Group
Sourced in United States
Nitroblue tetrazolium (NBT) dye is a chemical compound used in various laboratory applications. It is a blue-colored dye that is commonly used as an indicator for the detection and visualization of certain enzymatic activities, particularly those involving the reduction of oxygen. NBT dye is widely used in various fields of research, including cell biology, biochemistry, and immunology.
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2 protocols using nitroblue tetrazolium nbt dye
1
Superoxide Release Measurement Assay
2
Assay for Superoxide Anion in AM Cells
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This assay was used to determine the production of super-oxide anion in AM cells. AM cells from BAL were grown in a 96-well plate in serum-containing medium at 37°C for 4 hours. We added 1 μg/mL nitroblue tetrazolium (NBT) dye (Sigma, St Louis, MO, USA) to each well. The cells were incubated at 37°C for 1 hour or until color developed. The dye is yellow and gives a blue color formazan product upon reduction by superoxide. The reaction was terminated by adding 100 μL of stop solution (10% DMSO; 10% SDS in 50 mM HEPES buffer). The plate was left at room temperature overnight for complete dissolution of formazan, and absorbance at 560 nm was recorded using a multiscan plate reader to quantify the concentration of superoxide anion. Triplicates were done for each sample and control.26 (link)
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