Nitro-blue tetrazolium (NBT) assay

The NBT assay was performed as described in Secombes (Isolation of salmonid macrophages and analysis of their killing activity. In: Techniques in Fish Immunology) and Chettri (Factors influencing in vitro respiratory burst assays with head kidney leucocytes from rainbow trout, Oncorhynchus mykiss (Walbaum)).

After isolating cells from the head kindey, a cell suspension with 1*10^6 cells was added to 96 well plates and incubated over night to allow adherence of the cells.

Then cells were added either directly a NBT solution (1 mg/mL) or in addition to a stimulans (zymosan and PMA in our case but cell stimulation cocktails can be used as well to stimulate the respiratory burst). After 90 min of incubation, cells were fixed with 100 % Mehtanol and washed with 70 % Methanol. After drying, the resulting pelett of formazan (formed by conversion of NBT by reactive oxygene species) was dissolved in 2 M KOH and DMSO and the optical density was measured at 620 nm.