Serum lysozyme activity was measured with a turbidimetric method
on a subsample of 13 LPS-treated and 15 control animals [26,27]. Stored
haemolymph was thawed on ice and centrifuged at 13,000 g for 10 min
at 4 °C. Fifty μl of supernatant was mixed with 150 μl of substrate, i.e.
freeze driedMicrococcus lysodeikticus (Sigma; 0.075 g in 100 ml of citrate
phosphate buffer: Na2HPO4·2H2O 25 mM; citric acid 14 mM; pH 5.8) in
sterile 96 well flat bottom plate. The absorbance at 450 nm was determined
immediately after the addition of substrate and after 5 min
incubation at room temperature in a microplate reader. Fifty μl of citrate
phosphate buffer added to 150 μl of substrate served as blank solution.
Results were expressed as units/ml, one unit being defined as
the amount of lysozyme that causes a decrease in absorbance of
0.001 min−1 in the experimental conditions described above [28].
The immunological parameters, analysed before the immunetreatment
(T = 0 h), were again measured 4 h (T = 4 h) and 24 h
(T = 24 h) after the immune-treatment. All octopuses were reweighted
24 h after the treatment