Optimising flow-cytometry methods for marine mollusc haemocytes using the pearl oyster Pinctada maxima as a model

Flow-cytometry has become increasingly popular to assess the haemocytes morphology and functions of marine molluscs. Indeed, haemocytes are the first line of defence of the immune system in molluscs and are used as a proxy for oyster health. Authors publishing in the field of flow-cytometry and molluscs health seemed to utilise the same methods for all model species used, independently of their geographical location in the world (temperate, tropical, etc.). Hence, this paper dived into flow-cytometry methodology and investigated if using different plates, different thresholds, different incubation times and temperatures as well as different fluorochromes concentrations affected the results. This study revealed that the cell count did not change when using different thresholds on the FSC-H parameter of the instrument but was affected by the plate type, the temperature of incubation, and the time of incubation. Indeed, non-adherent plates yielded the highest cell count and lower cell counts were associated with a higher temperature and a longer time of incubation. Furthermore, the haemocytes functions such as the phagocytosis, the lysosomal content, the intracellular oxidative activity, and the mitochondria activity were also affected by the temperature and the time of incubation. An increase in the phagocytosis capacity, lysosomal content and mitochondria activity was observed with a higher temperature. At the exception of the phagocytosis rate, all the other parameters such as the phagocytosis capacity, the intracellular oxidative activity, and the lysosomal content increased with a longer incubation time. We also showed that it is best to optimise the amount of fluorochromes used to avoid unnecessary background or non-specific staining.