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

2.4. Isolation of head kidney phagocytes
Five specimens of soles of 200 g mean weight were sacrificed by an overdose of
clave oil and head kidney phagocytes to test respiratory burst activity were isolated
following the technique described by Secombes [32]. Briefly, the kidney was removed
aseptically and pushed through a 100 μm nylon mesh with Leibovitz medium (L-15)
containing 2% foetal bovine serum (FBS, Sigma), 1% penicillin-streptomycin (Sigma),
0.1% (5 mg ml -1 ) gentamicine sulfate (Sigma) (P/S/G) and 10 U heparine ml -1 . This cell
suspension was layered on a 30 to 51% Percoll (Amersham) gradient and centrifuged at
600 xg for 30 min, without brake. Then, the bands separated at the interface were
collected, centrifuged for 15 min at 500 xg 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 22ºC, non-adherent cells were removed and medium was
substituted by L-15 and P/S/G supplemented with 2%FBS. Monolayers were incubated
overnight at 22ºC, before use.
2.5. Respiratory burst activity
The generation of intracellular superoxide radicals by sole phagocytes, in
response to in vitro contact with algal polysaccharide, was determined by the reduction
of nitro-blue tetrazolium (NBT) according to the technique described by Secombes [32]
and Boesen et al. [33]. Volumes of 20 μl containing 1, 2, 5 and 10 mg ml -1 of the
lyophilized polysaccharidic fraction from P. cruentum were added to the wells
containing phagocyte monolayers obtained as described above. The response of sole
phagocytes to β-glucan from Euglena gracilis was also evaluated and 20 μl of serial
dilutions containing 1, 2, 5 and 10 mg ml -1 to 12 wells with sole phagocyte monolayers.
Response of sole phagocytes to the infection with P. damselae subsp. piscicida was
determined after inoculation of algal polysaccharide or β-glucan treated monolayers
with 20μl of bacterial suspensions containing 10 8 bacteria ml -1 . Phorbol myristate
acetate (PMA, Sigma) (1 μg ml -1 ) was used as a positive control to stimulate the
respiratory burst activity of sole phagocytes (data not shown). The specifity of the
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