Comparative study of β-glucan induced respiratory burst measured by nitroblue tetrazolium assay and real-time luminol-enhanced chemiluminescence assay in common carp (Cyprinus carpio L.)

• Published in: Fish & shellfish immunology 2013-05, Vol.34 (5), p.1216-1222
• Main Authors: Vera-Jimenez, N.I., Pietretti, D., Wiegertjes, G.F., Nielsen, M.E.
• Format: Article
• Language: English
• Subjects: Animals; beta-Glucans - metabolism; Carps - metabolism; Common carp; Head Kidney - metabolism; Hydrogen Peroxide - metabolism; Leukocytes - metabolism; Luminescence; Luminescent Measurements - methods; Luminol - metabolism; NBT assay; Nitroblue Tetrazolium - metabolism; Oxidants - metabolism; Real-time luminol assay; Respiratory Burst; Respiratory burst kinetics; Sensitivity and Specificity; Superoxides - metabolism; β-glucans
• ISSN: 1050-4648; 1095-9947
• DOI: 10.1016/j.fsi.2013.02.004

The respiratory burst is an important feature of the immune system. The increase in cellular oxygen uptake that marks the initiation of the respiratory burst is followed by the production of reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide which plays a role in the clearance of pathogens and tissue regeneration processes. Therefore, the respiratory burst and associated ROS constitute important indicators of fish health status. This paper compares two methods for quantitation of ROS produced during the respiratory burst in common carp: the widely used, single-point measurement based on the intracellular reduction of nitroblue tetrazolium (NBT) and a real-time luminol-enhanced assay based on the detection of native chemiluminescence. Both assays allowed for detection of dose-dependent changes in magnitude of the respiratory burst response induced by β-glucans in head kidney cells of carp. However, whereas the NBT assay was shown to detect the production of only superoxide anions, the real-time luminol-enhanced assay could detect the production of both superoxide anions and hydrogen peroxide. Only the chemiluminescence assay could reliably record the production of ROS on a real-time scale at frequent and continual time intervals for time course experiments, providing more detailed information on the respiratory burst response. The real-time chemiluminescence assay was used to measure respiratory burst activity in macrophage and neutrophilic granulocyte-enriched head kidney cell fractions and total head kidney cell suspensions and proved to be a fast, reliable, automated multiwell microplate assay to quantitate fish health status modulated by β-glucans. ► The paper compares NBT assay and RT-luminol assay as tools to monitor ROS in carp. ► RT-luminol assay allows tracking of kinetics during the respiratory burst response. ► RT-luminol assay detects the production of H2O2 and oxygen related radicals. ► RT-luminol assay can be used as a tool to monitor ROS involved in wound healing.