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

2.2. Alga culture
The red microalga Porphyridium cruentum (S.F. Gray) Näegli was obtained
from the collection at the Centro de Investigaciones Marinas de Cádiz, Cádiz, Spain. It
was grown in Porphyridium medium [30] in batch culture at 25ºC, with a 12h
photoperiod for 7 days. The algal biomass was centrifuged at 3000 xg, 15 min at 4ºC
and the pellet was lyophilized.
2.3. Obtention of aqueous and ethanolic extracts
The preparation of water-soluble extract, aqueous extract, was carried out as
follows: 10 g of lyophilized alga was resuspended in 100 ml of HBSS (Hank’s Balanced
Salt Solution) using a mortar and pestle. The extract was sonicated for 20 min and
centrifuged at 3000 xg, 5 min. The supernatant was separated from the pellet and
lyophilized and 10 mg of the lyophilized extract was resuspended in 1 ml of HBSS.
Extraction of the non-soluble fraction of the alga, ethanolic extract, was carried out as
described above, but instead of HBSS, ethanol was used. Dilutions from both extracts
were prepared in HBSS to achieve concentrations of 10 mg ml -1 of lyophilized extract, 5
mg ml -1 , 2 mg ml -1 and 1 mg ml -1 .
Commercial β-1,3-glucan from Euglena gracilis (BioChemika Fluka, Sigma)
was used as a positive control of stimulation of respiratory burst activity. β-1,3-glucan
(10 mg) was dissolved following commercial instructions, and diluted in HBSS to
achieve concentrations of 10 mg ml -1 , 5 mg ml -1 , 2 mg ml -1 and 1 mg ml -1 .
2.4. Fish and experimental design
Experiments to test the in vitro effect of P. cruentum on the respiratory burst
activity of sole phagocytes were carried out. Sole of 200 g body weight, stocked in 250 l
tanks with recirculating, aerated seawater at 20 ºC, 35‰ salinity, were used to isolate
kidney phagocytes and determine the production of anion radicals in contact with
aqueous and ethanolic extracts from P. cruentum and the commercial β-1,3-glucan.
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