Evaluation of fluorescence-based viability stains in cells dissociated from scleractinian coral Pocillopora damicornis

Abstract

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 molecules, proteins and nanoparticles (NP), was measured after 24 h 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 h to Triton-X100, insulin or titanium dioxide (TiO2) NPs, respectively, at concentrations ranging from 0.5 to 100 µg/mL, revealed a LC50 of 0.46 µg/mL for Triton-X100, 6.21 µg/mL for TiO2 NPs and 33.9 µg/mL for insulin. This work presents the approach used to customize dye pairs for membrane integrity-based cell viability assays considering the species- and genotype-specific autofluorescence of scleractinian corals, namely: endogenous fluorescence characterization followed by the selection of dyes that do not overlap with endogenous signals.

Plasmonic Au@Ag Core–Shell Nanoisland Film for Photothermal Inactivation and Surface-Enhanced Raman Scattering Detection of Bacteria

“…Once inside the cell, SYTOX green binds to DNA and emits green fluorescence when exposed to ultraviolet light or blue light [ 49 ]. Staining bacteria with SYTOX green and observing the green fluorescence under a microscope allows for the differentiation between live and dead bacteria on a surface [ 50 ]. Bacteria mortality was assessed by fluorescence imaging after exposure to light using SYTOX green staining (5 µM) for 30 min.…”

“…After the 360 min culture period under these elevated temperatures, the OD600 values of E. coli were only 0.10, 0.18, and 0.14, respectively. Furthermore, S. aureus was also exposed to varying temperatures (37,45,50,55, and 60 °C) to assess their impact on growth. As shown in Figure 3b, at the standard 37 °C culture temperature, S. aureus exhibited robust exponential growth, with an OD600 value reaching >2.0 after 360 min.…”