The respiratory burst activity of P. bocourti peripheral blood
leucocytes was determined using a modification of the method
described by Secombes [29]. 175 ml samples of 6 106 cells ml1 in
PBS were placed in the wells of 96-well microtiter plates. 25 ml of
Nitro Blue Tetrazolium (NBT) at a concentration of 1 mg ml1 was
added and incubated at 25 C for 2 h. The supernatant was carefully
discarded and then 125 ml of 100% methanol was added to each
well. Discard all supernatant and wash each well again with 125 ml
of 70% methanol well1 for twice. The supernatant in each well was
then carefully discarded and the plate was dried at room temperature
for 30 min. After that 125 ml of 2N KOH and 150 ml of DMSO
were added to each well. The plate was then measured at 655 nm
by a microplate reader (Sunrise, TECAN; Germany). Spontaneous
O2
production ¼ (Absorbance NBT reduction of sample)-
(Absorbance of blank).