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

Autor: Roger LM; Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, VA, USA. rogerlm@vcu.edu.; School of Molecular Sciences, Arizona State University, Phoenix, AZ, USA. rogerlm@vcu.edu., Adarkwa Darko Y; Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, VA, USA., Bernas T; Anatomy and Neurobiology, Virginia Commonwealth University, Richmond, VA, USA., White F; Anatomy and Neurobiology, Virginia Commonwealth University, Richmond, VA, USA., Olaosebikan M; Department of Computer Sciences, Tufts University, Boston, MA, USA., Cowen L; Department of Computer Sciences, Tufts University, Boston, MA, USA., Klein-Seetharaman J; School of Molecular Sciences, Arizona State University, Phoenix, AZ, USA.; College of Health Solutions, Arizona State University, Phoenix, USA., Lewinski NA; Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, VA, USA. nalewinski@vcu.edu.
Jazyk: angličtina
Zdroj: Scientific reports [Sci Rep] 2022 Sep 12; Vol. 12 (1), pp. 15297. Date of Electronic Publication: 2022 Sep 12.
DOI: 10.1038/s41598-022-19586-7
Abstrakt: 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 (TiO 2 ) 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 TiO 2 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.
(© 2022. The Author(s).)
Databáze: MEDLINE
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