Corals create biodiverse ecosystems important for both humans and marine organisms. However, we still do not understand the full potential and function of many coral cells. Here, we present a protocol developed for the isolation, labeling, and separation of stony coral cell populations.
Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the mechanisms are not well understood. To investigate coral response to stressors, we need tools for analyzing cellular responses. In particular, we need tools that facilitate the application of functional assays to better understand how cell populations are reacting to stress. In the current study, we use fluorescence-activated cell sorting (FACS) to isolate and separate different cell populations in stony corals. This protocol includes: (1) the separation of coral tissues from the skeleton, (2) creation of a single cell suspension, (3) labeling the coral cells using various markers for flow cytometry, and (4) gating and cell sorting strategies. This method will enable researchers to work on corals at the cellular level for analysis, functional assays, and gene expression studies of different cell populations.
Coral reefs are one of the most important ecosystems on Earth. They facilitate biodiversity by providing critical habitats for fish and invertebrates and are crucial for sustaining anthropogenic communities by providing food and economic livelihood through tourism1. As the key builder of coral reefs, the coral animal (Phylum: Cnidaria) also aids coastal communities by creating large calcium carbonate frameworks that mitigate wave and storm damage2.
Corals as adults are sessile animals that host a wide array of endosymbiotic partners, including viruses, archaea, bacteria, protists, fungi, and most notably, members of the algal dinoflagellate family Symbiodiniaceae3. Changes in the environment can cause imbalances in this community, often leading to disease outbreaks and coral bleaching in which the symbiotic Symbiodiniaceae are expelled from the coral colony, thus eliminating the major source of nutrition for the coral. Both of these scenarios often cause death of the coral host4,5,6. Effects of anthropogenic-induced stressors, such as rapid climate change, are accelerating mass coral death events, leading to a global decline of coral reefs7.
Recently, many different methods have been developedto help mitigate coral reef loss. These methods includeoutplanting of corals on existing reefs, genetic crossing using thermally tolerant genotypes, and cellular manipulation of the microbial and symbiotic communities hosted within the coral8,9. Despite these efforts, much remains unknown about coral cell diversity and cell function10,11,12,13. A thorough understanding of coral cell type diversity and cell function is necessary to understand how the coral organism behaves under normative and stressful conditions. Efforts to maximize restoration and preservation efficiency will benefit from an enhanced understanding of how cell diversity and gene function are coupled.
Previous work on cell diversity and function has primarily focused on histological studies and whole-tissue RNA sampling14,15,16,17. To obtain greater detail on specific cell type function in corals, there need to be methods for the isolation of specific populations of live coral cells. This has been done successfully in nonclassical model organisms by means of fluorescence-activated cell sorting (FACS) flow cytometry technology18. FACS utilizes a combination of lasers tuned to varying wavelengths to measure different endogenous cellular properties at the single cell level such as relative cell size, cell granularity, and autofluorescence. Additionally, the cells may be marked by fluorescently labeled compounds to measure specific, desired properties18,19.
Thus far, the application of flow cytometry to coral cells has mainly been for the analysis of symbiotic Symbiodiniaceae and other bacterial populations by utilizing their strong, natural autofluorescence20,21,22. FACS has also been used to estimate coral genome size by using fluorescent DNA marker signal compared against reference model organism cells23,24. The efficient application of FACS provides three distinct tools that are useful for cell biology studies: 1) morphological and functional description of single cells; 2) identification, separation, and isolation of specific cell populations for downstream studies; and 3) the analysis of functional assays at the single cell level.
The development and application of various exogenous fluorescent markers for the study of coral cells remains almost unexplored. Such markers may include tagged proteins, tagged substrates for enzymes, or fluorescent responses to other compounds. These markers can be used to identify cell types that have unique properties, such as highlighting cells that produce varying amounts of a specific cellular compartment feature, like lysosomes. An additional example is the use of fluorescently labeled beads to functionally identify cells competent for phagocytosis, or the engulfment of a targeted pathogen25. Populations of cells active in immunity responses can be easily identified by FACS after engulfment of these exogenously applied beads. While traditional histological methods require preserved tissue and many hours to approximate the percentage of cells positive for bead engulfment, a FACS-based functional assay for pathogen engulfment can be performed relatively quickly on isolated live cells. In addition to studying cell-specific responses to stress, this technology has the potential to clarify gene-specific expression and illuminate the evolutionary and developmental history of cell types entirely unique to cnidarians, such as calicoblasts and cnidocytes.
Recently, we performed an intensive screening of over 30 cellular markers that resulted in identifying 24 that are capable of labeling coral cells, 16 of which are useful for distinguishing unique populations18, making them clusters of differentiation (CD). Here we describe the process of coral cell isolation in Pocillopora damicornis from removing cells from the calcium carbonate skeleton to the identification and isolation of specific cell populations with FACS (Figure 1).
1. Dissociation of tissues from coral skeleton via airbrush and compressor
NOTE: Perform steps on ice and protect hands with gloves.
2. Dissociation of cells from coral tissue
NOTE: Perform all steps on ice and protect hands with gloves.
3. Cell staining
NOTE: Perform all steps on ice and protect hands with gloves. Stains featured in this protocol are for representation purposes. Alternative stains will require different concentrations and incubation times.
4. FACS startup
NOTE: The steps may vary according to the make and model of the cytometer due to differences in the lasers and channels. For this protocol, a cytometer with 405, 488, 535, and 640 nm wavelength lasers was used. Filters featured in this protocol are for representation purposes. Alternative cell stains may require a different set of filters and lasers.
5. FACS gating setup
NOTE: Steps may vary according to make and model of the cytometer and the acquisition program coupled with the cytometer.
6. FACS analysis and cell isolation
NOTE: Steps may vary according to the make and model of the cytometer and the coupled acquisition program.
7. FACS sorting and collection
NOTE: Steps may vary according to make and model of the cytometer and the acquisition program coupled with the cytometer.
Overall, this protocol is useful because it facilitates the identification and collection of live coral cell populations that can be used for functional analyses. The workflow started with the mechanical separation of coral tissues from the underlying calcium carbonate skeleton (Figure 1). This is one of the most important initial steps because improper technique results in high cell mortality and can create large amounts of debris. Enzymatic separation is not advised because it can result in increased tissue shearing and high cell mortality. Mechanical separation using an airbrush reduces these challenges (Figure 2), reducing average cell mortality to ~10% (data not shown). Following airbrush-mediated mechanical separation of tissues from the skeleton, the resulting slurry was filtered to a cell suspension by filtration through a cell strainer. The cell suspension was then stained with DNA (DAPI), ROS, and lysosome markers and read by the FACS cytometer. A gating strategy was then developed for isolating specific cell populations based on the cell markers being used. Gated cell populations were then sorted into collection tubes (Figure 1).
When analyzing samples on the FACS cytometer, several steps were taken in order to remove debris and dead cells from the sample. Symbiodiniaceae cells could be selectively removed if desired due to their autofluorescence. First, debris and other noncellular particles that did not share the same shape or granularity as coral cells were removed from analyses by creating a gating selection on the forward (size) and side (granularity) scatter (Figure 3A). Next, dead cells were excluded by comparing the unstained control cell suspension against DAPI-stained sample and far-red channel, then gating out the positive cells for high DAPI staining in the stained sample (Figure 3B, cell population P6). In parallel, cells hosting autofluorescent Symbiodiniaceae were removed by gating against cells with a high signal in the far-red channel (Figure 3B, APC-Cy7). After this iterative gating process, the remaining cells for analyses represent the intact live coral cell populations lacking Symbiodiniaceae.
These cells could then be classified into different cell subpopulations by comparing the control and stained samples by adding exogenous stains for features such as ROS activity and/or lysosome content (Figure 3C). For example, examining the data on the ROS- and lysosome-staining filters revealed four distinct subpopulations of coral cells. One cell population had a relatively low signal for lysosomal content (APC filter) and the other three had high signal in the lysosomal content and a range of ROS activity (FITC filter) (Figure 3C). Each of these subpopulations can be individually sorted for downstream analysis, or further analyzed and parsed into more discrete subpopulations by the intersection of size, granularity, autofluorescence, and/or DNA content. In this study, DNA, ROS activity, and lysosomal content were used to identify broad characteristics of coral cells. Importantly, this general protocol can be adapted to a range of fluorescent markers as well as specific antibody probes that can be used in conjunction for the isolation of specialized cell populations of interest such as stem cells.
Figure 1: The general workflow of the coral cell sorting process. The featured protocol enables the removal of corals cells from the skeleton for staining, which are then run through the FACS cytometer, measured, analyzed, and isolated into cell populations. Please click here to view a larger version of this figure.
Figure 2: Removal of tissue and cells from the coral skeleton. The air compressor (A) forces air and cell staining media (F) out of the airbrush (E) and works to blast the tissue off the coral skeleton (C) and creates a cell slurry in the bag (D). The maximum air pressure is controlled by the pressure gauge (B) on the air compressor (A). Please click here to view a larger version of this figure.
Figure 3: Gating selection of live coral cells. (A) Live cells were isolated from debris using a minimum forward scatter value of 102. (B) The APC-Cy7 filter was used to identify red autofluorescence, effectively separating out the symbiont-hosted cells, while DAPI was used to identify cells highly positive for DNA staining. Those highly positive for DAPI were indicative of dead and dying cells. (C,D) Additional markers for ROS activity and lysosome content were used to further differentiate different coral cell populations. All data was later reanalyzed on FlowJo (v10) to generate these graphs. The inserted population label and percent corresponds to the microscopic photos taken of each sorted population (E). The percentages shown in each plot are of the total cells within that plot. Bottom right scale bar = 20 µm. Please click here to view a larger version of this figure.
Figure 4: Purity check of the autofluorescent Symbiodiniaceae-associated cells. (A) Cells positive for fluorescence on the APC-Cy7 filter from the unstained control sample were sorted. (B) This sorted group of cells was then rerun on the cytometer and reanalyzed under the same conditions, showing 94% purity. Forward scatter (X-axis, FSC) was used for the anchoring axis, but is interchangeable with any of the other parameters. The same process can be performed on stained samples. Please click here to view a larger version of this figure.
This protocol was adapted from Rosental et al.18 and developed for the identification and isolation of P. damicornis cells. The methodology focuses on the process of filtering samples to remove debris, nonviable cells, and Symbiodiniaceae-hosted cells through the examination of cell intrinsic factors, including relative cell size, relative cell granularity, cell autofluorescence, and the presence of intact cellular membranes. These techniques can be applied to other coral species. However, FACS gating strategies may vary due to species-specific differences in cell population characteristics.
The utility of FACS for understanding coral health lies in its ability to use diverse cell intrinsic factors to differentially identify and isolate cells. Further, this protocol describes the additional use of exogenous cellular staining to differentiate coral cells based on viability, reactive oxygen species concentration, and lysosome content. Of the 30+ commercial markers that were formerly tested in Rosental et al.18, 24 labelled P. damicornis cells, of which 16 produced differential signal, clusters of differentiation that would allow for the selection of cell subpopulations.
One of the critical steps of this protocol is proper tissue removal and filtering to ensure that tissue clumps are dissociated and do not block the laminar flow of the flow cytometer. Proper adjustment of the PMT voltage is crucial for the accurate detection and analysis of independent cell detection events. Compared to previous work18, the process of coral cell isolation from the underlying skeleton via airbrush mechanical disruption significantly improves upon traditional scraping methods by reducing debris and maximizing cell viability (~90%, Figure 2 and Figure 3A), thereby improving subsequent analyses by reducing false positives due to noise.
The methodology described in this paper facilitates the separation of coral cell populations through differential selection on an intersection of morphological and functional characteristics at a level not previously possible. With the development of FACS methodologies for coral cells, it is now possible to select specific cell subpopulations for the creation of defined cell cultures, as well as for probing transcriptomic and epigenetic profiles associated with specific cell subpopulations. The application of this fine-scale information will provide insight into cell type, behavior, and function, and may reveal characteristics associated with the evolution of cnidarian-specific cell types such as calicoblasts and cnidocytes. Additionally, the application of FACS technology to the development of improved stress assays and biomarkers may prove useful for the protection of severely threatened coral reefs.
The authors have nothing to disclose.
NTK would like to acknowledge the University of Miami Research Awards in Natural Sciences and Engineering for funding this research. BR would like to thank Alex and Ann Lauterbach for funding the Comparative and Evolutionary Immunology Laboratory. The work of BR was supported by Israel Science Foundation (ISF) numbers: 1416/19 and 2841/19, and HFSP Research Grant, RGY0085/2019. We would like to thank Zhanna Kozhekbaeva and Mike Connelly for technical assistance. We would also like to thank the University of Miami, Miller School of Medicine’s Flow Cytometry Shared Resource at the Sylvester Comprehensive Cancer Center for access to the FACS cytometer and to Shannon Saigh for technical support.
Airbrush Kit & Compressor | TCP Global | ABD KIT-H-SET | Paasche H Series Single-Action Siphon Feed Airbrush Kit with Master TC-20 Compressor & Air Hose |
BD FACSAria II | BD | 644832 | |
Bone Cutters | Bulk Reef Supply | 205357 | Oceans Wonders Coral Stony Bone Cutter |
Cell Strainer | Corning | 352340 | 40 um; BD Falcon; individually wrapped; sterile; nylon |
CellRox Green | Life Technologies | C10444 | 2.5 mM in DMSO; Excitation/Emission: 485/520 nm |
Collection bag | Grainger | 38UV35 | Reloc Zippit 6"L x 4"W Standard Reclosable Poly Bag with Zip Seal Closure, Clear; 2 mil Thickness |
DAPI | Invitrogen | D1306 | 10mg in H2O; Excitation/Emission: 358/461 nm |
Fetal Calf Serum | Sigma-Aldrich | F2442-100ML | Heat-inactivated at 57 °C for 30 minutes |
Hemacytometer | Sigma-Aldrich | Z359629 | Bright-Line Hemacytometer |
HEPES Buffer | Sigma-Aldrich | H0887 | |
LysoTracker Deep Red | Life Technologies | L12492 | 1mM in DMSO; Absorption/Emission: 647/668 nm |
Microcentrifuge tubes | VWR | 87003-294 | 1.7 mL |
Phophate Buffered Saline (PBS) | Gibco | 70011-044 | pH 7.4; 10X |
Round-bottom tubes | VWR | 352063 | 5 mL Polypropylene Round-Bottom Tube |
Syringe | BD | 309628 | 1 mL BD Luer-Lok Syringe sterile, singe use polycarbonate |