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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spreading an aliquot of the bacterin on TSA p<strong>la</strong>tes and incubation for 2 days at 22 ºC.<br />
The vaccine was administered by intraperitoneal injection (0.1 ml per fish). Control fish<br />
were injected with phosphate buffer saline (PBS, pH 7.2).<br />
2.6. Iso<strong>la</strong>tion of head kidney phagocytes<br />
Sole phagocytes were iso<strong>la</strong>ted from the kidney following the technique<br />
<strong>de</strong>scribed by Secombes [31]. Briefly, the kidney was removed aseptically and pushed<br />
through a 100 μm nylon mesh with Leibovitz medium (L-15) containing 2% foetal calf<br />
serum (FCS, Sigma), 1% p<strong>en</strong>icillin-streptomycin (Sigma), 0.1 % (5 mg ml -1 )<br />
g<strong>en</strong>tamicine (Sigma) (P/S/G) and 10 U heparine ml -1 . This cell susp<strong>en</strong>sion was <strong>la</strong>yered<br />
on a 30 to 51% Percoll (Amersham) gradi<strong>en</strong>t and c<strong>en</strong>trifuged at 600 xg for 30 min.<br />
Th<strong>en</strong> the bands separated at the interface were 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 22 ºC, 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% FCS. Mono<strong>la</strong>yers were incubated<br />
overnight at 22 ºC.<br />
2.7. Respiratory burst activity<br />
The g<strong>en</strong>eration of intracellu<strong>la</strong>r superoxi<strong>de</strong> radicals by sole phagocytes was<br />
<strong>de</strong>termined by the reduction of nitro-blue tetrazolium (NBT) according to the technique<br />
<strong>de</strong>scribed by Secombes [31] and Boes<strong>en</strong> et al. [32]. Phagocyte mono<strong>la</strong>yers were washed<br />
with L-15 medium and HBSS (Hank´s Ba<strong>la</strong>nced Salt Solution) to remove any trace of<br />
the antibiotic.<br />
In or<strong>de</strong>r to test the in vitro effects of algal extracts on the production of<br />
superoxi<strong>de</strong> radicals by sole phagocytes, a volume of 20 μl of the extracts (aqueous,<br />
ethanolic or β-glucan) were ad<strong>de</strong>d to the wells (15 wells from 5 fish) containing<br />
phagocyte mono<strong>la</strong>yers prepared as above. Th<strong>en</strong>, 100 μl of NBT dissolved at 1 mg ml -1<br />
in HBSS was ad<strong>de</strong>d to the wells and the phagocytes incubated at 22 ºC for 30 min.<br />
Wells containing phagocytes were infected with 20 μl of P. damse<strong>la</strong>e subsp. piscicida<br />
7