Optimization of cell culture protocols and immunoassays in a non-model teleost.

Abstract The threespine stickleback fish and its natural helminth parasite Schistocephalus solidus are an emerging model for studying host-parasite immune modulation. However, to serve as an effective immunological and host-pathogen interaction model in vitro protocols must be developed and optimized to assess immune function, including leukocyte viability, apoptosis, production of reactive oxygen species (ROS), phagocytosis, and proliferation. Unique features of stickleback leukocytes make them incompatible with many commercially available reagents and their small size make them undetectable by some instruments, presenting challenges in the development of these assays. Flow cytometry has proven to be a successful tool for performing streamlined cell counts and we have succeeded in identifying live, apoptotic, and dead leukocytes in this species. We have also optimized flow cytometry assays to detect the production of multiple ROS species using DHR-1,2,3 as well as a superoxide-specific detection assay. The small size of stickleback leukocytes makes them incompatible with large phagocytosis particles, such as zymosan. However, using a combination of lipophilic cell membrane dye and fluorescently tagged E. coli particles, in conjunction with confocal microscopy, we successfully quantified the phagocytic activity of stickleback splenocytes and confirmed internalization of the particles. Lastly, identifying proliferating stickleback leukocytes has been difficult; both EdU and Ki67 assays have been unsuccessful. Although challenging, identifying and documenting successful techniques to use with non-model organisms is essential for expanding our immunologic tool box and furthering our knowledge of these organisms. Supported by grants from NIH (R01 AI146168)