Papel de las actividades superóxido dismutasa y catalasa en la ...

separated at the interface were collected, centrifuged for 15 min at 500 × g and
resuspended in L-15 medium supplemented with P/S/G. The viable cell concentration
was determined after staining with trypan blue and microscope counting. Aliquots of
100 μl containing 1x10 7 cells ml -1 in L-15 medium supplemented with P/S/G were
added to 96-well microtitre plates. After 3 h incubation at 22 ºC, non-adherent cells
were removed and medium was substituted by L-15 and P/S/G supplemented with
2%FCS. Monolayers were incubated overnight at 22 ºC.
2.4. Respiratory burst activity
Fish of the four experimental groups, fed with diets A, B, C or D, were assayed
for 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 [45] and Boesen et al. [46]. Phagocyte monolayers were washed
with L-15 medium and HBSS (Hank´s Balanced Salt Solution) to remove any trace of
antibiotic. Then, NBT (100 μl) dissolved at 1 mg ml -1 in HBSS was added to the wells
and the phagocytes incubated at 22 ºC for 30 min. Wells containing phagocytes were
infected with P. damselae subsp. piscicida (10 8 bacterias ml -1 ) and used to determine the
response of the phagocytes to the fish pathogen. As a positive control phorbol myristate
acetate (PMA, Sigma) (1 μg ml -1 ), an activating agent of the respiratory burst, was used
to stimulate the respiratory burst of non-infected phagocytes. The specificity of the
reaction was tested by adding superoxide dismutase (SOD) (300 I.U. per well) to some
wells containing PMA-stimulated phagocytes (data not shown).
After incubation, cells were fixed in 70% methanol and reduced formazan within
phagocytes was solubilised by adding 120 μl 2M KOH and 140 μl dimethyl sulfoxide
(DMSO, Sigma). Finally, absorbance was read at 630 nm in a multiscan
spectrophotometer (UV-1601 Spectrophotometer, Whitakker Bioproducts).
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