A flow cytometric approach to study intracellular-free Ca2+ in Crassostrea gigas haemocytes.

Bivalve haemocytes are essential in defence mechanisms including phagocytosis. They also produce molecules including hydrolytic enzymes and antimicrobial peptides that contribute to pathogen destruction. Although haemocyte activities have been extensively studied, relatively little is known about the intracellular signalling pathways that are evoked during haemocyte activation and especially the role of calcium. Flow cytometry has been used for the first time to define the effect of cell incubation in haemolymph and artificial sea water (ASW) on Pacific oyster, Crassostrea gigas, haemocytes. Cell viability, enzymatic activities (esterases and aminopeptidases), phagocytosis and granulocyte percentage were analysed. Viability and some activities were different in haemolymph and ASW. Cytoplasmic-free calcium in circulating haemocytes was then investigated by flow cytometry in both media using a calcium probe (Fluo-3/AM). To explore calcium homeostasis, different calcium modulators were tested. The calcium chelator Bapta/AM (10 microM) reduced significantly the percentage of Fluo-3-positive cells in ASW. In addition, ryanodine (5 microM) induced a significant enhancement of the percentage of Fluo-3 positive cells in haemolymph and in ASW. Flow cytometry may be used to study calcium movements in C. gigas haemocytes, but several haemocyte incubation media need to be tested in order to confirm results. The objective of the study should be considered before selecting a particular experimental medium.