Nitroblue Tetrazolium Assay(NBT) for estimation of superoxide anion in phagocytic cells

The NBT assay is used to estimate the ability of cells to produce reactive oxygen species(ROS). This assay is mainly used in immunology for the semiquantitative measurement of ROS produced inside the phagocytic cells. In this report, we will briefly understand how NBT is used for the estimation of superoxide anion in phagocytic cells and the mechanism lying behind it.

How is ROS formed inside phagocytic cells?

NADPH oxidase which is a membrane bound enzyme present in phagocytic cells especially macrophages and neutrophils gets activated when there is an entry of pathogen. It leads to the transfer of electrons from NADPH to oxygen resulting in the production of ROS such as superoxide anion. This phenomenon is known as respiratory burst.

NADPH + 2O₂ ↔ NADP⁺ + 2O₂⁻ + H⁺

How ROS kills pathogen?

ROS is one of the most important antimicrobial components by immune cells but the actual mechanism of ROS killing pathogen is still unclear. When the respiratory burst occurs in macrophage it leads to signal transductions such as pathogen recognition receptor signaling, As a result, several microbes killing cells get activated and perform processes like autophagy, lymphocyte proliferation, etc

What is Nitroblue Tetrazolium?

NBT is a pale yellow in color chemical compound composed of two tetrazole moieties. When NBT interacts with ROS it is reduced to a blue coloured precipitate called formazon. It is commonly used in clinics to diagnose chronic granulomatous disease (CGD), an immunologic disease caused by a reduced oxidative burst resulting in a limited ability to kill phagocytosed bacteria.

Chemicals used in NBT assay and its function:

1. Trypan blue: it is a dye which stain dead tissues blue in colour. It is negatively charged so it interacts with membrane only when cell membranes are damaged. As undamaged cells are very selective about what type of compound is entering the membrane so it does not get stain with trypan blue.

2. DMEM (Dulbecco’s modified eagle medium): it is a basal medium for supporting the growth of many different mammalian cells (fibroblasts, neurons, etc). It is basically a modification of basal medium eagle so it contains a higher concentration of amino acids, vitamins, and other additional components.

3.Lipopolysaccharide(LPS): It is present in the outer membrane of gram negative bacteria and so can be used as a pathogen to activate macrophage.

4.1,4-dioxane: it is a heterocyclic organic compound that helps in dissolving blue coloured formazan.

5. Phosphate buffered saline (PBS): it is a buffer solution which helps in maintaining a constant pH. The osmolarity and ion concentration of the solution is kept isotonic in order to match the human body. It mainly consists of sodium chloride, potassium chloride, etc.

How is NBT assay performed?

1. In order to perform NBT assay the first crucial step is the isolation of the phagocytic cells for example macrophages. It is done by collecting the required cells under buffer condition with the help of PBS and centrifuging it to take the pellet and dissolve it in DMEM.
2. The 2nd step is to check the total number of viable cells with the help of trypan blue which is known as viability assay. The counting is done with the help of a hemocytometer.
3. Finally, the phagocytic cells are mixed with NBT after the addition of the LPS. After proper incubation for a few minutes, 1 to 2 drops of HCl are added and later centrifugation is performed. 1,4-dioxane is added to the pellet obtained from centrifugation. After incubation optical density is measured in 520nm as the absorbance of the formazan blue colored solution is measured in this wavelength in a spectrophotometer.

How result is interpreted?

The reduction of NBT to formazon takes place only when the mitochondrial reductase enzymes are active and therefore conversion can be directly related to the number of viable cells. When the amount of formazan produced is treated with an agent, here LPS is compared with the amount of formazan produced by untreated control cells it helps in inferring the number of death of cells occurred.

Conclusion

This assay has been widely used for the determination of activated phagocytes by different methods. However, the technical limitations of these methods have been evaluated carefully and observed that the conventional microscopic NBT assay has numerous limitations. Thus the modified colorimetric NBT assay is sensitive, quantitative, and convenient, and is able to detect even the small amounts of O2 produced by weak O2 producers.

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Reference

Müller, J., Alföldy, P. and Lemmel, E.M., 1981. Nitroblue-tetrazolium test for the functional evaluation of phagocytic cells: A critical analysis of the methodology. Agents and actions, 11(4), pp.384-390.