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

(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 ) was
used to stimulate the respiratory burst activity of non-infected phagocytes (data not
shown). 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 the 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).
In the P. cruentum feeding experiments, the effect of oral administration of the
alga or Sanostim was determined on phagocyte monolayers as described above, but
algal extracts or bacteria were not added to the wells.
2.8. Statistical analysis
Results are expressed as the stimulation index (mean + standard error, SE),
obtained by dividing each sample value by the mean control value. Values higher than 1
reflect an increase and lower than 1 a decrease in each parameter compared to the
control. Data were statistically analysed by one-way analysis of variance (ANOVA) and
Tukey’s comparison of means using SPSS for Windows. Differences were considered
statistically significant when P< 0.05.
3. Results
3.1. In vitro assays
Results have been expressed as the ratio of the absorbance at 630 nm of treated
phagocytes to the absorbance of non-treated phagocytes. The treatment of phagocytes
consisted of incubation with aqueous (Figure 1a) or ethanolic (Figure 1b) algal extracts
or β-glucan (Figure 1c), in the presence or absence of P. damselae subsp. piscicida.
Therefore the data represented in the Figures are the result of a correlation between
phagocytes incubated with the algal extract or β-glucan, with or without bacteria, and
phagocytes incubated without the potential immunostimulant (algal extract or β-glucan).
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