Evaluation of fluorescence-based viability stains in scleractinian coral cell cultures

The application of established cell viability assays such as the commonly used trypan blue staining method to coral cells is not straightforward due to different culture parameters and different cellular features specific to mammalian cells compared to marine invertebrates. Using Pocillopora damicornis as a model, we characterized the autofluorescence and tested different fluorescent dye pair combinations to identify alternative viability indicators. The cytotoxicity of different representative molecules, namely small organic molecule, protein and nanoparticles (NP), was measured after 24 hours of exposure using the fluorescent dye pair Hoechst 33342 and SYTOX® orange. Our results show that this dye pair can be distinctly measured in the presence of fluorescent proteins plus chlorophyll. P. damicornis cells exposed for 24 hours to Triton-X100, insulin or titanium dioxide (TiO2) NPs, respectively, at concentrations ranging from 0.5-100 µg/mL, revealed a LC50 of 0.5 µg/mL for Triton-X100, 20 µg/mL for TiO2 NPs and an average 20% reduction in viability at 100 µg/mL for insulin. The workflow presented here provides a general framework for customizing dye pairs for cell viability assays considering the species- and genotype-specific autofluorescence of scleractinian corals.