Papel de las actividades superóxido dismutasa y catalasa en la ...
Papel de las actividades superóxido dismutasa y catalasa en la ...
Papel de las actividades superóxido dismutasa y catalasa en la ...
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2.4. Iso<strong>la</strong>tion of head kidney phagocytes<br />
Five specim<strong>en</strong>s of soles of 200 g mean weight were sacrificed by an overdose of<br />
c<strong>la</strong>ve oil and head kidney phagocytes to test respiratory burst activity were iso<strong>la</strong>ted<br />
following the technique <strong>de</strong>scribed by Secombes [32]. Briefly, the kidney was removed<br />
aseptically and pushed through a 100 μm nylon mesh with Leibovitz medium (L-15)<br />
containing 2% foetal bovine serum (FBS, Sigma), 1% p<strong>en</strong>icillin-streptomycin (Sigma),<br />
0.1% (5 mg ml -1 ) g<strong>en</strong>tamicine sulfate (Sigma) (P/S/G) and 10 U heparine ml -1 . This cell<br />
susp<strong>en</strong>sion was <strong>la</strong>yered on a 30 to 51% Percoll (Amersham) gradi<strong>en</strong>t and c<strong>en</strong>trifuged at<br />
600 xg for 30 min, without brake. Th<strong>en</strong>, the bands separated at the interface were<br />
collected, c<strong>en</strong>trifuged for 15 min at 500 xg and resusp<strong>en</strong><strong>de</strong>d in L-15 medium<br />
supplem<strong>en</strong>ted with P/S/G. The viable cell conc<strong>en</strong>tration was <strong>de</strong>termined after staining<br />
with trypan blue and microscope counting. Aliquots of 100 μl containing 1x10 7 cells ml -<br />
1 in L-15 medium supplem<strong>en</strong>ted with P/S/G were ad<strong>de</strong>d to 96-well microtitre p<strong>la</strong>tes.<br />
After 3 h incubation at 22ºC, non-adher<strong>en</strong>t cells were removed and medium was<br />
substituted by L-15 and P/S/G supplem<strong>en</strong>ted with 2%FBS. Mono<strong>la</strong>yers were incubated<br />
overnight at 22ºC, before use.<br />
2.5. Respiratory burst activity<br />
The g<strong>en</strong>eration of intracellu<strong>la</strong>r superoxi<strong>de</strong> radicals by sole phagocytes, in<br />
response to in vitro contact with algal polysacchari<strong>de</strong>, was <strong>de</strong>termined by the reduction<br />
of nitro-blue tetrazolium (NBT) according to the technique <strong>de</strong>scribed by Secombes [32]<br />
and Boes<strong>en</strong> et al. [33]. Volumes of 20 μl containing 1, 2, 5 and 10 mg ml -1 of the<br />
lyophilized polysaccharidic fraction from P. cru<strong>en</strong>tum were ad<strong>de</strong>d to the wells<br />
containing phagocyte mono<strong>la</strong>yers obtained as <strong>de</strong>scribed above. The response of sole<br />
phagocytes to β-glucan from Eugl<strong>en</strong>a gracilis was also evaluated and 20 μl of serial<br />
dilutions containing 1, 2, 5 and 10 mg ml -1 to 12 wells with sole phagocyte mono<strong>la</strong>yers.<br />
Response of sole phagocytes to the infection with P. damse<strong>la</strong>e subsp. piscicida was<br />
<strong>de</strong>termined after inocu<strong>la</strong>tion of algal polysacchari<strong>de</strong> or β-glucan treated mono<strong>la</strong>yers<br />
with 20μl of bacterial susp<strong>en</strong>sions containing 10 8 bacteria ml -1 . Phorbol myristate<br />
acetate (PMA, Sigma) (1 μg ml -1 ) was used as a positive control to stimu<strong>la</strong>te the<br />
respiratory burst activity of sole phagocytes (data not shown). The specifity of the<br />
6