**"Evaluation of fluorescence-based vaibility stains in scleractinian coral cell cultures"**
Authors: Liza M. Roger1*, Yaa Adarkwa Darko1, Tytus Bernas2, Frances White2, Judith Klein-Seetharaman3,4, Nastassja A. Lewinski1*
1Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, VA 2Anatomy and Neurobiology, Virginia Commonwealth University, Richmond, VA 3School of Molecular Sciences, Wexford 310F, Arizona State University, Phoenix, AZ 4College of Health Solutions, Wexford 310F, Arizona State University, Phoenix, AZ *corresponding authors: rogerlm@vcu.edu and nalewinski@vcu.edu
**Supplementary Materials, S.1**: Spectral scans performed on Pocillopora damicornis live cells with seven different lasers (Zeiss LSM 710, VCU Microscopy Core):
405 nm (A)
440 nm (B)
458 nm (C)
488 nm (D)
514 nm (E)
561 nm (F)
633 nm (G)
**Software required to read these files: Zen 3.2 or higher.**
**Abstract**
The application of established cell viability assays to coral cells is not straightforward due to different culture parameters and different cellular features specific to mammalian cells compared to marine invertebrates. Issues related to the commonly used trypan blue staining method have been documented (binds to free enzymes in culture medium). Using Pocillopora damicornis as a model, we characterized the autofluorescence and tested different fluorescent dye pair combinations to identify alternative viability indicators. As a proof-of-concept, the cytotoxicity of titanium oxide nanoparticles (TiO2 NPs) and insulin 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 concentrations of TiO2 NPs or insulin between 0.5-100 µg/mL revealed a LC50 of 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 framework for customizing dye pairs considering the species- and genotype-specific autofluorescence of scleractinian corals.
**Keywords:** coral cell, viability, cytotoxicity, fluorescence, insulin, titanium oxide
**Microscopy Methods**
Single optical sections of coral cell suspension were acquired with a Zeiss LSM 880 or LSM 710 confocal microscope, build on Axio Observer Z1 inverted stand, equipped with motorized stage and a 40x Plan Apo oil immersion objective (NA 1.4). Fluorescence was excited with either one or a combination of lasers available on the system: 405 nm diode (15 mW), 440 nm diode (15 mW), multiline Ar ion (458/488/514 nm, 25 mW), 561 nm DPSS (15 mW), 594 nm and 633 nm He-Ne (3 mW each). Excitation power was adjusted between 0.8% and 2%, depending on the line. Fluorescence spectrum was registered with a 32-channel spectral hybrid detector at the gain of 500V. Each channel corresponded to 7.9 nm. The pinhole was set to 1.25 Airy units at 550 nm emission. Transmitted light (DIC) was registered together with fluorescence. Images were collected in tile-based mode (5x5), with total area of 1220 x 1220 um, 0.13 um pixel size and 2.05 us dwell time. All signals were digitized with 16-bit precision.
