2.10. Nitroblue Tetrazolium (NBT) Reduction Assay

To detect reactive oxygen species (superoxide anion (O²⁻), hydrogen peroxides (H₂O₂), and hydroxyl radicals (OH⁻) production by phagocytic cells), oxidative burst activity was measured by reduction of NBT by the method of Das et al. [33] with minor modification.

The optimum concentration of rLrIL-10 required to induce NBT reduction was determined in a preliminary experiment. Heparinized blood was collected from five fish. Blood samples (100 µL) from each fish (in triplicate) were treated with equal volume of PBS or rLrIL-10 (1000 ng in 100 µL PBS) and allowed to incubate for 1 h at 25°C. Equal volume of 0.2% NBT (Sigma-Aldrich Chemical Co., USA) was then added to the samples and incubated further for 1 h at 25°C to allow formazan granules formation. Intracellular reduction of NBT was determined by solubilizing the formazan crystals with dimethyl formamide (SRL, India), centrifugation at 2000 ×g for 5 min, and measurement of absorbance of the supernatant at 540 nm.