2.8.2. Respiratory burst activity
The generation of intracellular superoxide radicals by sole
phagocytes was determined by the reduction of nitro-blue tetrazolium
(NBT) according to the technique described by Secombes
[28] and Boesen et al. [29]. Phagocyte monolayers were washed
with L-15 medium and HBSS (Hank’s Balanced Salt Solution) to
remove any trace of antibiotics. Then, 100 ml NBT, dissolved at
1 mg ml1 in HBSS, were added to the wells and the phagocytes
incubated at 22 C for 30 min. After incubation, the medium was
removed and the culture was washed twice with isotonic PBS, fixed
with 100 ml/well of 100% methanol for 3 min, and washed twice
with 70% methanol; then, the cells were air dried. Formazan was
solubilized in 120 ml of KOH (2 M) plus 120 ml of dimethyl sulfoxide
(DMSO) and the absorbance was read spectrophotometrically
(Hitachi) at 620 nm using KOH/DMSO as blank.