Name: fluorescence-activated cell sorting for the isolation of scleractinian cell populations
Uploaded: 2020-05-31T12:30:00
Description: Watch this Scientific Journal Video about Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations at JoVE.com

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.&#160;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&#8217;s Flow Cytometry Shared Resource at the Sylvester Comprehensive Cancer Center for access to the FACS cytometer and to Shannon Saigh for technical support.

1. Dissociation of tissues from coral skeleton via airbrush and compressor
NOTE: Perform steps on ice and protect hands with gloves.
<ol>
	<li>Assemble the airbrush kit by connecting the air compressor, hose, and airbrush (Figure 2). Set the pressure gauge between 276&#8722;483 kPa. 
	NOTE: The recommended compressor and air hose used for this study was preset to a maximum pressure of 393 kPa. Use outside of this 276-483 kPa range may result in either inade...

<ol>
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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...

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).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Airbrush Kit &#38; Compressor</td>
			<td>TCP Global</td>
			<td>ABD KIT-H-SET</td>
			<td>Paasche H Series Single-Action Siphon Feed Airbrush Kit with Master TC-20 Compressor &#38; Air Hose</td>
		</tr>
		<tr>
			<td>BD FACSAria II</td>
			<td>BD</td>
			<td>644832</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone Cutters</td>
			<td>Bulk Reef Supply</td>
			<td>205357</td>
			<td>Oceans Wonders Coral Stony Bone Cutter</td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>Corning</td>
			<td>352340</td>
			<td>40 um; BD Falcon; individually wrapped; sterile; nylon</td>
		</tr>
		<tr>
			<td>CellRox Green</td>
			<td>Life Technologies</td>
			<td>C10444</td>
			<td>2.5 mM in DMSO; Excitation/Emission: 485/520 nm</td>
		</tr>
		<tr>
			<td>Collection bag</td>
			<td>Grainger</td>
			<td>38UV35</td>
			<td>Reloc Zippit 6&#34;L x 4&#34;W Standard Reclosable Poly Bag with Zip Seal Closure, Clear; 2 mil Thickness</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>10mg in H2O; Excitation/Emission: 358/461 nm</td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>Sigma-Aldrich</td>
			<td>F2442-100ML</td>
			<td>Heat-inactivated at 57 &#176;C for 30 minutes</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629</td>
			<td>Bright-Line Hemacytometer</td>
		</tr>
		<tr>
			<td>HEPES Buffer</td>
			<td>Sigma-Aldrich</td>
			<td>H0887</td>
			<td></td>
		</tr>
		<tr>
			<td>LysoTracker Deep Red</td>
			<td>Life Technologies</td>
			<td>L12492</td>
			<td>1mM in DMSO; Absorption/Emission: 647/668 nm</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>VWR</td>
			<td>87003-294</td>
			<td>1.7 mL</td>
		</tr>
		<tr>
			<td>Phophate Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>70011-044</td>
			<td>pH 7.4; 10X</td>
		</tr>
		<tr>
			<td>Round-bottom tubes</td>
			<td>VWR</td>
			<td>352063</td>
			<td>5 mL Polypropylene Round-Bottom Tube</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD</td>
			<td>309628</td>
			<td>1 mL BD Luer-Lok Syringe sterile, singe use polycarbonate</td>
		</tr>
	</tbody>
</table>

fluorescence-activated cell sorting for the isolation of scleractinian 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.

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 no...

Watch this Scientific Journal Video about Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations at JoVE.com

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

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 ...

This protocol facilitates the identification and isolation of live coral cell populations at a descriptional level not previously possible. The main advantage of this technique is that it allows access to a level of cell specificity that up until now has been mostly unattainable. Demonstrating the procedure will be Dr.Shannon Saigh, a research associate from the flow cytometry shared resource at the Sylvester Comprehensive Cancer Center.To set up the flow cytometer for live coral cell sorting, open a new project template in the flow cytometer software and select the appropriate lasers for the analysis in the laser panel. Then, create a side scatter versus forward scatter plot in the appropriate plots for setting up the gating and analysis strategy. For flow cytometric analysis and sorting of the live coral cells, load the control unstained cell sample into the sample chamber of the cytometer and start the reading process.Since coral cells are particularly fragile, keep the cytometer pressure low to prevent cell lysis. In the scatter plot, adjust the photo multiplier voltage to center the points and begin recording the events. In the forward versus side scatter plot, create a gate around the cells at around the 10 to the 4th mark on the forward scatter x-axis.Replace the control sample with the first sample of stained cells and record approximately 10, 000 cells before pausing. Then gate the events that are unique to the stained sample group. To sort live coral cells, after selecting the cell population of interest in the flow cytometer software, place one microcentrifuge tube containing 250 to 500 microliters of the appropriate collection solution into the cytometer collection chamber for each population to sort.Once an appropriate amount of time has passed to flush out debris, begin sorting the desired population to collect anywhere from 10, 000 to several million cells. Each time a new stain, species, or sorter is used, sort at least 20, 000 cells of interest into 500 microliters of staining medium to allow a purity check to be performed on the population of interest and reanalyze the sorted cells to confirm that the events are being read within the gate used for the initial sorting. For in vitro studies, store the sorted cells on ice until they're transferred to an incubator or sterilized environment.To bring in other non-cellular particles that do not share the same shape or granularity as the coral cells can be removed from the analysis by creating a gate selection on a forward and side scatter plot. The dead cells can be excluded by comparing the DAPI signal of the unstained and stained cell suspensions using a histogram and gating out the high DAPI-expressing cells. In parallel, cells hosting autofluorescence symbiodeneasia can be removed by gating against cells with the high signal in the far red channel.After this iterative gating process, the remaining cells represent the intact live coral cell populations lacking symbiodeneasia. These cells can then be classified into different cell subpopulations by staining for features such as reactive oxygen species activity and/or lysosome content. It's critical to remember that some cells are more fragile than others and to run this process as carefully as possible.Once specific cells have been isolated, they can be used for x-vivo functional assays such as establishing cell cultures and creating transcriptomic profiles.

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