IMMUNE CHALLENGE AND HARVESTING BLOOD FROM SEA URCHINS

When required, sea urchins were challenged with 1.2 x 108heat-killedE. colicells (inoculum volume of 0.2 ml) per animal for24 h or 48 h (for subsequent protein extraction and microscopy) prior to harvesting coelomic fluid.

Between 0.5 ml and 2.5 ml of coelomic fluid was harvested from each animal and mixed with an equal volume of ice-cold calcium- and magnesium-free sea water (CMFSW-EI; 460 mM sodium chloride, 10.73 mM potassium chloride, 7.04 mM disodium sulphate, 2.38 mM sodium hydrogen carbonate) with 30 mM EDTA and 50 mM imidazole, pH 7.4. Coelomocytes were pelleted (1200–5000 x g for 4 min at 4oC) and washed in CMFSW-EI before further processing.

Sham Nair 2014