Coral cell separation and isolation by fluorescence-activated cell sorting (FACS)
© The Author(s). 2017
Received: 15 November 2016
Accepted: 20 August 2017
Published: 29 August 2017
Generalized methods for understanding the cell biology of non-model species are quite rare, yet very much needed. In order to address this issue, we have modified a technique traditionally used in the biomedical field for ecological and evolutionary research. Fluorescent activated cell sorting (FACS) is often used for sorting and identifying cell populations. In this study, we developed a method to identify and isolate different cell populations in corals and other cnidarians.
Using fluorescence-activated cell sorting (FACS), coral cell suspension were sorted into different cellular populations using fluorescent cell markers that are non-species specific. Over 30 different cell markers were tested. Additionally, cell suspension from Aiptasia pallida was also tested, and a phagocytosis test was done as a downstream functional assay.
We found that 24 of the screened markers positively labeled coral cells and 16 differentiated cell sub-populations. We identified 12 different cellular sub-populations using three markers, and found that each sub-population is primarily homogeneous. Lastly, we verified this technique in a sea anemone, Aiptasia pallida, and found that with minor modifications, a similar gating strategy can be successfully applied. Additionally, within A. pallida, we show elevated phagocytosis of sorted cells based on an immune associated marker.
In this study, we successfully adapted FACS for isolating coral cell populations and conclude that this technique is translatable for future use in other species. This technique has the potential to be used for different types of studies on the cellular stress response and other immunological studies.
Coral reefs to coral cells
Reef building corals are extremely important organisms for marine ecosystems. Corals are diploblastic cnidarians that live in shallow, warm tropical regions, and are critical contributors to reef ecosystems that host much of the ocean’s biodiversity [1, 2]. They provide billions of dollars in revenue for coastal and island countries; however, despite this great importance they are under tremendous threat from anthropogenic climate change [3, 4]. Previous estimates have found that 32.8% of corals assigned to International Union for Conservation of Nature (IUCN) are in danger of extinction . Given their economical and environmental importance, it is imperative to gain a better understanding of how corals react to climate change.
It is well understood that corals will be under great stress under the projected climate change conditions . Reef building corals have an obligate symbiosis with a symbiotic dinoflagellate, Symbiodinium, and this relationship is one of the most well characterized cellular interactions in coral cell biology. When stressed this relationship breaks down and disrupts the intracellular relationship of Symbiodinium and its coral host. This process is called bleaching and it further contributes to the coral host’s stress.
Previously, many coral cellular studies have focused on coral host uptake of the Symbiodinium [6, 7], the breakdown of the coral host-Symbiodinium relationship [8–16], cellular calcification mechanisms [17–25], cell culture techniques [26–29], and the identification of the intracellular pH relationship between coral host cells and Symbiodinium [30–32]. Additionally, flow cytometry has been used to quantify Symbiodinium cells, and assay for apoptosis [33, 34]. Lastly, many cellular studies on corals have focused on the histological aspects of stress response and disease of the whole organism [35, 36].
However, other than the breakdown of the relationship between Symbiodinium and coral host cell during heat induced stress, little is understood about the role of other cell types during the cellular stress response. Previous studies have found that other cell types including cnidocytes, a family of stinging cell types found only in cnidarians, and other gastrodermal cells may be critical for the heat induced stress response in corals . Additionally, there is little information on the presence of immune-like cells called amoebocytes, in the scleractinian (stony or hard) corals. Previous characterization of amoebocytes was done in the gorgonian coral, Swiftia exserta, a non-scleractinian coral , and in scleractinian corals, amoebocytes have been identified by histology . In order to address these gaps, we have developed a protocol that uses fluorescence-activated cell sorting (FACS) to efficiently sort cells into different populations based on natural fluorescence and fluorescent cell dyes, allowing us to collect them for further analysis.
Coral cell types
Corals have two tissue layers, an outer epidermis and internal gastrodermis. These tissue layers are separated by a mesoglea, which harbors multiple cell types including secretory, amoeboid, and reproductive cells . Many cell types reside within the epidermis including ciliated column, secretory, sensory motor neuron, interneuron, neurosecretory, sensory cells, cnidocytes, and flagellated columnar cells . The cell types in the gastrodermis include cuboidal, absorptive, secretory, squamous, columnar, anchoring, flagellated columnar, flagellated cuboidal, spindle shaped, sensory cells, motor neurons, interneurons, neurosecretory and Symbiodinium (algal cells which live within the coral gastrodermal cells) . In addition to the endosymbiotic algal cells, there is also some evidence for endosymbiotic bacteria that live within coral tissue layers, however little is understood about their role and function in the coral .
Fluorescence-activated cell sorting (FACS)
Flow cytometry is a powerful technique used to distinguish and characterize cell types, including live cells. This technique, which has been used primarily in biomedical and immunological research, utilizes lasers to analyze and sort different cell types in real time based on specific properties of the cell. Applications of FACS include clinical analysis, cell purification, functional assays, and pathogen detection [41–47]. Although these techniques have not been widely applied to many non-medical systems, they are a powerful methods for cell type discovery and cell activity in comparative and evolutionary research. Furthermore, isolation of different cells, based on general properties (e.g. lectins, enzymes, size and granularity) that are not antibody- based can successfully be used in separating different cell populations in non-model species and these distinct cell populations are different functionally and physiologically [48–51].
Here, we have developed a method to separate coral cell populations by utilizing cell markers that are non- species specific. This powerful technique allows for cellular differentiation in real time. Using this technique a number of cellular functions can be measured including free radical production, immune properties, intracellular enzymatic activity, and compound uptake. This technique can also be used to separate specific cell populations for gene expression studies, which will allow for more targeted studies of the coral stress response. In this report, we present the methods of cell sorting, and strategies for distinguishing specific coral cells, and specific cell markers for characterizing coral cell types. Additionally, we tested this technique on a symbiotic anemone, Aiptasia pallida, as confirmation that this sorting strategy can be applied to other species. Lastly, we tested phagocytosis, as a downstream functional assay on sorted cells expressing high levels of lysolitic vesicles.
Coral fragment collection and coral cell dissociation
Fragments of Pocillopora damicornis were obtained from the Monterey Bay Aquarium (MBA) in partnership with the Tropical Coral Propagation program. These corals, which had previously been obtained from illegal shipments, were confiscated by the U.S. Fish and Wildlife Service and donated to MBA for propagation and research.
To prepare the coral cell suspension for FACS analysis, P. damicornis fragments of 1–4 cm in length were scraped off of the soft tissue with fine blade into a petri dish containing staining media: 3.3× PBS (without Ca and Mg), 2% FCS (vol/vol) and 20 mM Hepes, pH 7.4. The soft tissue in the petri dish was dissociated with a fine blade into cell suspensions and filtered through a 40 μm mesh. Using the plunger of a sterile 1 ml syringe, cell suspensions were additionally dissociated on the mesh, and washed and collected in staining media. The entire process took approximately 10 min per fragment and was done on ice to minimize damage to the cells and lower their metabolism. Cells were then washed by centrifugation at 500 x g at 4 °C for 5 min. Cells were counted using a hemocytometer and trypan blue diluted 1:10 (ThermoFisher Sientific), a cell viability dye that excludes dead cells. The trypan blue was diluted 1:10 (compared to mammalian cells usage) to 0.04%, due to aggregate formation at high salinity.
These same methods were then tested in a strain of A. pallida containing Symbiodinium and a aposymbiotic A. pallida strain (strain that is raised without Symbiodinium). These strains were kindly provided by the J. R. Pringle mariculture facility at Stanford University .
Cell labeling, phagocytosis and FACS analysis
Screened compounds to differentiate coral cells
Company and Catalog
Differ. Cells (Symb.+)
Arachidonoyl ethanolamide uptake
Cytochrome P450 activity
NBD-FTY720 phenoxy HCl
Aerobic metabolic activity
Coumarin Boronic Acid pinacolate ester
Histone acetyltransferase inhibitor
Intracellular Zinc ion
Intracellular Chloride ion
Binds α-man, α-glc
Binds α-L-fuc and (glcNAc)2
Binds β-gal(1 → 3)galNAc
Binds sialic acid and Nac
Intracellular pH indicator
LysoTracker Deep Red
Reactive oxygen species
Heavy metal indicator
AT regions of DNA
For the phagocytosis assay, we used green colored beads called Fluoresbrite® YG Carboxylate Microspheres 1.00 μm (Polysciences), in a 2:1 beads:cells ratio. Sorted cells were incubated for 3 h or overnight at room temperature in staining media (105cells/200 μl). The FACS reading was done either on BD FACS Aria II, BD LSRFortessa, BD FACS Accuri C6 (Becton, Dickinson Biosciences) or SH800S Cell Sorter (Sony). Analysis of flow cytometry data was done with FlowJo V10 program (FlowJo). The specific excitation laser and optical filter for emission measurements for each of the analysis is stated in the figures. The excitation laser was measured in nm and filters are stated as long pass (LP) and band pass (BP).
Cell sorting using FACS and live cells microscopy
Cells were sorted by using the BD FACS Aria II, and gating was done using the BD FACSDiva™ software (Becton, Dickinson Biosciences). Cell sorting was performed with 100 μm nozzle size and sorted directly into 5 ml tubes containing 3 ml of staining media in order to minimize cellular stress. Cells (10,000–40,000) of each population of interest were sorted at a speed of 1500 cells/s.
For live cells microscopy, after washing in staining media by centrifugation at 4 °C; 500 x g; 5 min, the cells were resuspended in 30 μl of staining media and placed onto 18 well flat μ-slides coated with poly-L-lysine (Ibidi) and incubated overnight at 4 °C. Images of sorted cells were obtained using light microscopy.
Cellular dissociation of coral cells and initial gating strategy
Dissociation of cells was accomplished using mechanical tools rather than enzymatic tools in order to prevent proteolysis of the membrane-bound proteins and to preserve cell viability. Using physical dissociation less cell death was observed, whereas, the use of the serine protease, trypsin, increased the percentage of dead cells in suspension by four-fold (Additional file 1: Figure S1).
Fluorescent cell markers for cellular differentiation
When screening for cell detection markers we looked for the following parameters: i) is there labeling of cells or not (positive staining compared to unstained control, Fig. 2b), ii) does the labeling enable cell population separation (Fig. 2d), and iii) if there is no separation, is there a wide dynamic range of fluorescence between high and low labeled cells that will distinguish different cell populations (Fig. 2c). For instance, labeling with Phen Green, which is used to identify metal ions, all the cells compared to the control were labeled, but a distinction between cell populations that express positive and those that express a higher absolute positive signal can be detected (marked in red, Fig. 2b). Another example is when using ALDEFLUOR to detect stem cells and pluripotent cells through the activity of aldehyde dehydrogenase (ALDH) . Labeling with ALDEFLUOR does not differentiate populations, but because the labeling is so broad (wide dynamic range: 103–106 in 640 nm (675/25BP) fluorescence channel, and 102–105 in the 488 nm (533/30BP) fluorescence channel) gates can be made to distinguish between low, moderate, and high fluorescent labeling (marked in red, Fig. 2c).
In the testing for reactive oxygen species (ROS) using CellROX and phagocytosis using microbeads, we found a clear positive population compared to the rest of the cells (gated in Fig. 2d, f). This indicates that there are populations of cells that are acting similar to immune cells and can be detected by immune markers. Glucose analog uptake measured by 2-NBDG, which is an indicator of the energetic state of the cells, is positive in all cells compared to unstained samples, but it does not give a clear signal of cell separation (Fig. 2e). Due to this reason the reagent was not a good candidate for identifying different cell types. Lastly, cells containing Symbiodinium can be differentiated by using the Far-Red [640 nm (675/25BP) channel (gated in Fig. 2a and Additional file 1: Figure S2)], and also together with the screened markers as seen in all panels of Fig. 2 in the Far-Red [640 nm (675/25BP)] channel.
Use of fluorescent cell markers for cell population sorting
We validated morphologically that we could sort different cell populations using the screened fluorescent markers. We then used a panel of three dyes (DAPI, Cellrox, and LysoTracker Deep Red) with a different channels gating strategy and sorted 12 coral cell populations (Fig. 4). The sorted cells were imaged in order to assess the morphology and homology within each sorted population (Image inserts in Fig. 4). Based on this panel, we succeeded in separating three populations of Symbiodinium positive cells that look very different at a morphological level (Fig. 4). In addition, 9 populations were sorted, which looked relatively homogeneous. For example, population 14 (P14) is enriched for cnidocytes, the stinging cells of cnidarians (Fig. 4d). This validated the assumption that the cells we sorted using our FACS differentiation markers and gating strategy were different cell populations on the morphological level.
Corals have two tissue layers, an outer epidermis and an internal gastrodermis. These layers are separated by a mesoglea layer, a primitive type of mesoderm, that is made of connective tissue, and has few cells present including secretory cells, amoeboid cells, and reproductive cells . Many cell types reside within the epidermis, including ciliated column cells, secretory cells, sensory motor neuron cells, interneuron cells, neurosecretory cells, sensory cells, cnidocytes, and flagellated columnar cells . In the gastrodermis, cell types include cuboidal cells, absorptive cells, secretory cells, squamous cells, columnar cells, anchoring cells, flagellated columnar cells, flagellated cuboidal cells, spindle shaped cells, sensory cells, motor neurons, interneurons, neurosecretory and Symbiodinium (algal symbiotic cells which live within gastrodermis cells) . Overall, in Fig. 4, we can see many of these previously mentioned cell types, including the cnidocytes in population P14, and Symbiodinium in populations P6, P7, and P8. The other cell populations appear to be a mix of various cells from each layer, but have similar fluorescent outputs, and therefore partitioned into the same population. Based on morphological observations, populations P13, and P5 appear to have very similar cell types present, including round granular type cells, and some rod shaped cells. Population P10 and P4 are also very similar; both have irregular shaped, smaller cell types. Population P15 has several cell types that have a granular appearance, as does population P3 and P1. Further investigation will need to be explored to characterize these cell types in this isolated form, and assign them to their tissue layer. One possibility is using the same labels with fluorescent microscopy on an intact specimen to try to understand the localization of the cells within the animal.
Validation of the fluorescent cell markers on symbiotic anemone
Validation of down-stream process on sorted cells based on lysolitic vesicles
Practical uses for live coral cell separation
In the current study we used mechanical dissociation, and discerned between live and dead cells and debris using FSC, SSC and DNA dyes. We then screened for different fluorescent markers to isolate and sort coral cell populations. We used a diverse array of flow cytometers for our analysis in order to show that this method can be done on variety of FACS instruments, from machines with 4 channels and 2 lasers (BD Accuri C6; Figs. 2 and 5) to machines with 14 channels and 4 lasers (BD LSRFortessa; Fig. 3). We also developed cell isolation methods for coral tissue, and identified conditions in which cells can be cultured ex-vivo. Lastly, we assayed for ex-vivo cellular function, such as phagocytosis and glucose uptake (Figs. 2, 5 and 6).
These methods have many important down-stream applications, including cell culture, cell specific functional assays, RNA-sequencing, immune assays, transplantation, as well as gene and protein expression done in mammalian and non-mammialian animal models [43–45, 48, 49, 51]. As a proof of concept, we performed phagocytosis assays on sorted cell populations (Fig. 6). Utilizing this technique in corals will allow for a better understanding of different coral cells types, and will be useful in understanding how different cell populations react to different stressors, including heat-induced stress, immune challenges, endosymbiosis processes, and acidification treatments, In total, we screened over 30 different cell markers, which can be used to identify different cellular populations. In the future, a combination of our described FACS methods using the same reagents for fluorescent microscopy in a gross animal specimen could reveal localization of specific cell populations in-vivo.
Identification of different coral cell populations
We examined the use of fluorescent markers to separate coral cell populations. From this, we identified 12 different populations including three that include Symbiodinium (Fig. 4). We have shown that we can use a combination of screened dyes to sort large numbers of cellular populations with different morphologies. We would like to emphasize that we separated cells based on three non-species specific markers. Combinations of other markers, or the addition of specific antibodies, will lead to a greater degree of population separation. The markers that we have used are associated with a specific enzymatic or cellular activity, a compound, or an organelle, and therefore are indicators of biological function. For example, cells positive for ROS and LysoTracker (Population 13 in Fig. 4) could be immune-like phagocytic cells; we would expect this type of cell to have lysolitic vesicles containing ROS compounds [55–58]. These cells could be targeted for future research using beads and bacteria engulfment to demonstrate immune-like phagocytosis. In A. pallida, we have successfully shown that LysoTracker positive cells are associated with higher phagocytosis level compared to LysoTracker low cells (Fig. 6).
The ALDH high population may also prove to be a population of interest (Fig. 2c, gated in upper right side). High ALDH activity is associated with stem and progenitor cells  and can enrich stem cells in invertebrates . Isolation of these cells would be valuable for understanding the regenerative capabilities of corals and could lead to a better understanding of wound healing processes.
Lastly, we found markers that did not clearly label different cell populations, such as the glucose uptake marker 2-NBDG (Fig. 2e). While it is not a good marker for cell separation, it is a good candidate for assaying glucose uptake in corals in experimental settings such as in response to heat stress. Previous studies have shown that glucose and glycerol are translocated from Symbiodinium to the coral host . These critical metabolites are important for the coral overall health and low levels could indicate a nutritional deficit for the coral host.
While the majority of the markers used today in flow cytometry are antigen-specific, we have used non-species specific fluorescent labeling compounds that can be easily translated to non-model organisms. As a proof of concept, we performed assays on two different cnidarians and found labeling patterns to be very similar (Fig. 5 compared to Fig. 2). Therefore, it is likely that this method could be used on other cnidarian species. Corals express many different fluorescent proteins and there is variation in expression between different corals . Therefore, minor adjustments may be necessary in developing a gating strategy for the successful separation in these populations. With the validation of this technique in both A. pallida and P. damicornis, we are confident that this strategy will be valuable for studies in other cnidarians as well.
In this study, we adapted the FACS method for studying coral cells. We developed a gating strategy for identifying 12 separate coral cell populations and tested over 30 different fluorescent markers. We verified this in another cnidarian, A. pallida, and found that a similar strategy can be used, indicating the utility of this technique in other cnidarians. We have shown that sorted cells can be used in down-stream live functional assays and that the markers used provide valuable insights into the biology of coral cells. This technique will be extremely valuable for studying the cellular role of stress response, as well as for immunological studies in corals and other cnidarians.
We would like to thank Dr. Stephen R. Palumbi of Stanford University for lab space, Raymond Direen of the Monterey Bay Aquarium for providing the coral samples, and Dr. Varghese K. George and Dr. Savita Pahwa for assistance with this project. We thank Dr. Lorraine Ling and Dr. John R Pringle for kindly providing A. pallida strains. We would also like to thank Karla J. palmeri, Dr. Jody M. Beers, and Paul A. K. Bump for editing. This work was funded by National Science Foundation Ocean Science Postdoctoral Fellowship OCE-1323652 and award #1012629 from the Burroughs Wellcome Postdoctoral Enrichment Fund for N.T.K. The work of B.R. was supported by the Postdoctoral Fellowship of the Human Frontier Science Program Organization and the NIH Hematology training grant T32 HL120824-03.
BR and NTK designed the study. BR conducted the study. BR, NF and JMT designed the screening chemical compounds panels. BR and ZK validated the sorting and cell isolation. BR and NTK wrote the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Fragments of Pocillopora damicornis were obtained from the Monterey Bay Aquarium (MBA) in partnership with the Tropical Coral Propagation program. These corals, which had previously been obtained from illegal shipments, were confiscated by the U.S. Fish and Wildlife Service and donated to MBA for propagation and research. A. pallida and also an aposymbiotic A. pallida strain (strain that is raised without Symbiodinium) was kindly provided from J. R. Pringle mariculture facility at Stanford University.
Consent for publication
The authors declare that they have no competing interests.
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