Name: Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations (Video) | JoVE Journal
Uploaded: 2020-05-31T12:30:00.000+00:00
Duration: 4 min 32 s
Description: Learn fluorescence-activated cell sorting to isolate live scleractinian coral cell populations with high specificity. This protocol enables functional assays and transcriptomic studies for marine biology research.

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5 minutes

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 inadequate cell removal from the skeleton or rupture of the cell membranes. This range may differ for each species and may require a viability test. A viability test may be performed with a subset of the cell slurry with a simple hemocytometer and a 4&#8242;,6-diamidino-2-phenylindole (DAPI) dye exclusion assay, visualized on a compound microscope.</li>
	<li>Prepare 100 mL of cell staining media in an autoclaved, sterile glass container by adding 33 mL of 10x phosphate-buffered saline (PBS; without calcium and magnesium) to a final concentration of 3.3x, 2 mL of fetal calf serum (FCS; heat inactivated at 56 &#176;C for 30 min) to 2% of total volume, and 2 mL of 1 M HEPES buffer to a final concentration of 20 mM. Then top off at 100 mL with sterile water. 
	NOTE: The 3.3x PBS concentration was chosen to mimic the salinity of seawater, as demonstrated in Rosental et al.18. This concentration needs to be adjusted for species found in other environments to mimic their salinity.</li>
	<li>Transfer 10 mL of cell staining media to an airbrush bottle (labeled &#34;F&#34; in Figure 2) and attach the adaptor lid for the airbrush connector. 
	NOTE: Larger fragments and species with thicker tissue layers may require more media for adequate tissue removal.</li>
	<li>For the branching coral species demonstrated here, use a bone cutter to clip or cut a coral fragment approximately 3&#8722;5 cm in length or 4 cm2 in area. 
	NOTE: The fragment must be clear of macroalgae and other multicellular organisms because these may not be removed from the sample downstream and will contaminate the sample during the FACS analysis and sorting processes. A 1 cm fragment of P. damicornis gives approximately 2&#8722;10 x 106 cells after filtering, staining, and washing.</li>
	<li>Place the coral fragment inside a plastic collection bag and use the airbrush to spray the cell staining media onto the coral (Figure 2). Continue this process until most of the skeleton is exposed. Remove the remaining skeleton from the collection bag.</li>
</ol>
2. Dissociation of cells from coral tissue
NOTE: Perform all steps on ice and protect hands with gloves.
<ol>
	<li>Filter the cell slurry through a 40 &#181;m nylon cell strainer into a 50 mL centrifuge tube in order to obtain a single cell suspension. 
	NOTE: Different cell strainer mesh sizes may be more appropriate for different species. However, larger sizes may result in inadequate tissue dissociation due to the passage of debris and cell clumps.
	<ol>
		<li>For specimens with thicker mucus layers, use the sterilized plunger of a 1 mL plastic syringe and gently grind it against the filter to break up clumps and help the cells pass through the strainer. Rinse the strainer and plunger with cell staining media into the centrifuge tube.</li>
	</ol>
	</li>
	<li>Pellet the cells by centrifugation at 4 &#176;C at 450 x g for 5 min. Remove the supernatant and resuspend the cells in 1 mL of cell staining media.</li>
</ol>
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.
<ol>
	<li>Transfer 500 &#181;L of resuspended cells to a 5 mL round-bottom tube and set aside as a control sample. Do not add stain to this aliquot.</li>
	<li>To the remaining cell suspension, add 0.42 &#181;L of 12 mM DAPI viability dye (Table of Materials), 1 &#181;L of 5 &#181;M reactive oxygen species (ROS) stain (Table of Materials), and 0.1 &#181;L of 0.2 &#181;M lysosome stain (Table of Materials). Pipette well to mix and incubate on ice for 30 min in the dark to prevent photobleaching. 
	NOTE: DAPI is used in this example to work as a DNA stain and viability dye for the differential gating of dead cells on the FACS machine. This assumes that DAPI penetrates dying cells due to membrane integrity. The ROS stain emits light in the 520 nm range (green), while the lysosomal marker emits light at approximately 668 nm (red).</li>
	<li>Pellet 500 &#181;L of the stained cells by centrifugation at 4 &#176;C at 450 x g for 5 min. Remove the supernatant and resuspend the pellet in 500 &#181;L of cell staining media. Transfer to a new 5 mL round-bottom tube and store on ice.</li>
</ol>
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.
<ol>
	<li>Begin creating a new project template on the sorter software and choose the laser panel. For the represented combination of stains and natural fluorescence, select all four lasers (405, 488, 535, and 640 nm).</li>
	<li>Select the appropriate filters for each expected color represented in the experiment.
	<ol>
		<li>To detect the DAPI emission and aid in the separation of live and dead cells, use a filter with a bandpass (BP) of 450/50 (425&#8722;475 nm range) such as a DAPI, Hoeschst, or Pacific Blue filter.</li>
		<li>For the detection of light emitted by the green ROS signal, use a filter with a BP of 530/30 (wavelength range of 515-545 nm) such as a fluorescein isothiocyanate (FITC) or green fluorescent protein (GFP) filter. This filter will measure the concentration of ROS in each cell.</li>
		<li>Add an additional filter with a BP of 670/30 (655&#8722;685 nm range) such as an allophycocyanin (APC) filter for the detection of the lysosomal stain emission. 
		NOTE: The APC channel will allow for the measurement of phagocytic activity. This channel will also detect the autofluorescence generated by the coral symbiotic algae from the family Symbiodiniaceae. This signal can be separated using an additional channel that has a longer wavelength than the APC filter, however.</li>
		<li>Include a filter that has a BP of 780/60 (750-810 nm range), such as the most commonly used phycoerythrin, cyanine filter (APC-Cy7), which detects the emission of far-red autofluorescence from Symbiodiniaceae and aids in the isolation of aposymbiotic cells.</li>
	</ol>
	</li>
</ol>
5. FACS gating setup
NOTE: Steps may vary according to make and model of the cytometer and the acquisition program coupled with the cytometer.
<ol>
	<li>On the project experimental screen of the cytometry software, create a scatter plot and select forward scatter (FCS) as the metric for the X-axis and side scatter (SSC) for the Y-axis. 
	NOTE: FCS correlates with the size of the cell and SSC correlates with granularity. This will allow for the removal of most debris, as most will be smaller in size than intact cells.</li>
	<li>Set the axes to either logarithmic or biexponential scales, as cell sizes and granularity may vary by several orders of magnitude (Figure 3A), particularly in corals.</li>
</ol>
6. FACS analysis and cell isolation
NOTE: Steps may vary according to the make and model of the cytometer and the coupled acquisition program.
<ol>
	<li>Place the control (i.e., the unstained cell subset) in the sample chamber of the cytometer and start the reading process. When the computer starts to receive data from each cell, or event, dots will begin to appear on the scatter plot. To clear any debris left in the cytometer chambers from previous experiments, allow approximately 30 s to pass before starting the analysis.</li>
	<li>In the scatter plot, adjust the photomultiplier tube (PMT) voltage in order to center the points. 
	NOTE: The PMT voltage is used to amplify the signal strength of photons being measured; if the PMT voltage is too weak, fewer cells will be read, and if the PMT voltage is too strong, cells will be measured near maximum value and different cell types will be indistinguishable from one another. An adequate PMT voltage ensures that independently distinguishable events will populate the scatter plot. Event separation is easier to visualize by projecting logarithmic or biexponential scales on FSC and SSC scatter plots.</li>
	<li>Start recording the events. To conserve the sample, pause data acquisition after approximately 15,000 events have been read. In most cases, this will be adequate to visualize the overall cell population patterns. Record more events if analyzing and isolating small, specialized cell populations.</li>
	<li>On the first graph of FSC and SSC, create a selection, or gate, of the cells around the 102 mark and higher on the FSC X-axis. Anything below this threshold is likely cellular debris. Draw rectangular gates, which is a regular option on flow cytometry software programs and good for clear, distinct cell populations. For cell populations that take a more irregular shape on the scatter plots, use a polygonal gating option. 
	NOTE: A minimum cutoff of 102 on the forward scatter should select for the smallest coral cells but may allow contamination of debris. To amend this, increase the lower limit of the gating to be more conservative.</li>
	<li>Create a new scatter plot expressing the DAPI filter on the X-axis and the APC-Cy7 filter on the Y-axis. To do this, select the gate for intact cells created in step 6.4, right-click, and select the option to create a new scatter plot. Adjust the axes to either logarithmic or biexponential scales. 
	NOTE: The steps needed to create a new plot may vary with different software programs.</li>
	<li>Now remove the control sample from the cytometer chamber and replace with the stained cells. Allow 30 s to pass before starting the analysis and recording the data. Record approximately 15,000 cells before pausing. 
	NOTE: The distinct population(s) on the higher end of the APC-Cy7 scale are those with high red autofluorescence from Symbiodiniaceae. No stain is needed to visualize these population breaks, and they can also be viewed on the control sample recording (Figure 3C). Select for the coral-only populations, those lower on the Y-axis, for further differentiation.</li>
	<li>Create a new scatter plot of the aposymbiotic cells to visualize the ROS concentrations (FITC filter) and the cells positive to lysosomes (APC filter), again utilizing logarithmic or biexponential scales. 
	NOTE: If there are multiple distinct cell populations that are aposymbiotic, each one can be further distinguished in its own scatter plot (Figure 3C,D).</li>
	<li>Compare the stained sample group against the control, unstained subset by either using the first recording of the control sample or reinserting and rerunning the control group. Gate the events that are unique to the stained sample group.</li>
</ol>
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.
<ol>
	<li>With a selection of cells chosen to collect, place microcentrifuge tubes (containing 250&#8722;500 &#181;L of cell culture media or lysis buffer based on number of cells collected and method of storage) in the cytometer collection chamber. 
	NOTE: The represented flow cytometer is capable of sorting four different populations at a time, and the machine can be configured to hold a variety of collection devices, including various centrifuge tubes and 96 well plates.</li>
	<li>Once the cytometer starts to read the sample and an appropriate amount of time has passed to flush out debris, start sorting the desired populations and collect anywhere from 20,000 cells to several millions.
	<ol>
		<li>For more than 500,000 cells, adjust the volume of cell culture media or lysis buffer, and the volume of the collection device.</li>
		<li>For in vitro studies, store cells on ice until placed in an incubator or sterilized environment.</li>
		<li>For molecular studies, either immediately start the DNA/RNA/protein isolation process or flash freeze and store at -80 &#176;C until extraction.</li>
	</ol>
	</li>
	<li>Each time a new stain, species, or sorter is used, perform a purity check on the population of interest by sorting at least 20,000 cells of interest into 500 &#181;L of staining media, then re-analyzing the sorted cells and confirming that the cells are being read within the gate used to initially sort (Figure 4).</li>
</ol>

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<li>Hoegh-Guldberg, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coral+reefs+under+rapid+climate+change+and+ocean+acidification%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Coral reefs under rapid climate change and ocean acidification.</a> Science. 318 (5857), 1737-1742 (2007).</li>
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<li>Peters, E. C. Anatomy. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPeters%2C+EC+%22Diseases+of+Coral%22">Diseases of Coral</a>. Woodley, C. M., et al. , Wiley Blackwell. Boston, MA. 85-107 (2015).</li>
<li>Allemand, D., Tambutté, É, Zoccola, D., Tambutté, S. Coral calcification, cells to reefs. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aAllemand%2C+D+%22Coral+Reefs%3A+An+Ecosystem+in+Transition%22">Coral Reefs: An Ecosystem in Transition</a>. Dubinsky, Z., Stambler, N. , Springer Netherlands. Dordrecht, Netherlands. 119-150 (2011).</li>
<li>Rinkevich, B., Loya, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Reproduction+of+the+Red+Sea+Coral+Stylophora+pistillata.+I.+Gonads+and+Planulae%5BTitle%5D)+AND+%22Marine+Ecology+Progress+Series%22%5BJournal%5D)">The Reproduction of the Red Sea Coral Stylophora pistillata. I. Gonads and Planulae.</a> Marine Ecology Progress Series. 1 (2), 133-144 (2007).</li>
<li>Palmer, C. V., Traylor-Knowles, N. G., Willis, B. L., Bythell, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Corals+use+similar+immune+cells+and+wound-healing+processes+as+those+of+higher+organisms%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Corals use similar immune cells and wound-healing processes as those of higher organisms.</a> PLoS ONE. 6 (8), e23992(2011).</li>
<li>Hayes, R. L., Bush, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microscopic+observations+of+recovery+in+the+reef-building+scleractinian+coral%2C+Montastrea+annularis%2C+after+bleaching+on+a+Cayman+reef%5BTitle%5D)+AND+%22Coral+Reefs%22%5BJournal%5D)">Microscopic observations of recovery in the reef-building scleractinian coral, Montastrea annularis, after bleaching on a Cayman reef.</a> Coral Reefs. 8 (4), 203-209 (1990).</li>
<li>Chapman, D. M. Cnidarian Histology. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aChapman%2C+DM+%22Coelenterate+Biology%22">Coelenterate Biology</a>. Muscatine, L., Lenhoff, H. M. , Academic Press. New York, NY. 1-43 (1974).</li>
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<li>Traylor-Knowles, N., Palumbi, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Translational+environmental+biology%3A+Cell+biology+informing+conservation%5BTitle%5D)+AND+%22Trends+in+Cell+Biology%22%5BJournal%5D)">Translational environmental biology: Cell biology informing conservation.</a> Trends in Cell Biology. 24 (5), 265-267 (2014).</li>
<li>Rosental, B., Kozhekbaeva, Z., Fernhoff, N., Tsai, J. M., Traylor-Knowles, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coral+cell+separation+and+isolation+by+fluorescence-activated+cell+sorting+%28FACS%29%5BTitle%5D)+AND+%22BMC+Cell+Biology%22%5BJournal%5D)">Coral cell separation and isolation by fluorescence-activated cell sorting (FACS).</a> BMC Cell Biology. 18 (1), 1-12 (2017).</li>
<li>Rosental, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complex+mammalian-like+haematopoietic+system+found+in+a+colonial+chordate%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Complex mammalian-like haematopoietic system found in a colonial chordate.</a> Nature. 564 (7736), 425-429 (2018).</li>
<li>Silva-Lima, A. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiple+Symbiodinium+Strains+Are+Hosted+by+the+Brazilian+Endemic+Corals+Mussismilia+spp%5BTitle%5D)+AND+%22Microbial+Ecology%22%5BJournal%5D)">Multiple Symbiodinium Strains Are Hosted by the Brazilian Endemic Corals Mussismilia spp.</a> Microbial Ecology. 70 (2), 301-310 (2015).</li>
<li>Yvan, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+cytometric+enumeration+of+bacterial+in+the+coral+surface+mucus+layer%5BTitle%5D)+AND+%22Journal+of+Microbiological+Methods%22%5BJournal%5D)">Flow cytometric enumeration of bacterial in the coral surface mucus layer.</a> Journal of Microbiological Methods. 128, 16-19 (2016).</li>
<li>Lee, C. S., Wilson Yeo, Y. S., Sin, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bleaching+response+of+Symbiodinium+%28zooxanthellae%29%3A+Determination+by+flow+cytometry%5BTitle%5D)+AND+%22Cytometry+Part+A%22%5BJournal%5D)">Bleaching response of Symbiodinium (zooxanthellae): Determination by flow cytometry.</a> Cytometry Part A. 81 (10), 888-895 (2012).</li>
<li>Baumgarten, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+genome+of+Aiptasia%2C+a+sea+anemone+model+for+coral+symbiosis%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">The genome of Aiptasia, a sea anemone model for coral symbiosis.</a> Proceedings of the National Academy of Sciences. 112 (38), 11893-11898 (2015).</li>
<li>Voolstra, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+analysis+of+the+genomes+of+Stylophora+pistillata+and+Acropora+digitifera+provides+evidence+for+extensive+differences+between+species+of+corals%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Comparative analysis of the genomes of Stylophora pistillata and Acropora digitifera provides evidence for extensive differences between species of corals.</a> Scientific Reports. 7 (1), 1-14 (2017).</li>
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</ol>

The authors have nothing to disclose.

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><tbody><tr><td>Airbrush Kit &amp;amp; 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 &amp;amp; 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&quot;L x 4&quot;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 &amp;deg;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

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...

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 ...

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JoVE Journal

Biology

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

Fluorescence Activated Cell Sorting of Plant Protoplasts

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFRα as a surface marker.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

Isolation and Characterization of Single Cells from Zebrafish Embryos

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

This protocol describes customizable surface functionalization of the desthiobiotin, streptavidin, and APTES system in order to isolate specific cell types of interest. In addition, this manuscript covers the applications, optimization, and verification of this process.

A Method of Targeted Cell Isolation via Glass Surface Functionalization

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Flow cytometry in combination with visual clustering offers an easy-to-use and fast method for studying aquatic biofilms. It can be used for biofilm characterization, detection of changes in biofilm community structure, and detection of abiotic particles embedded in the biofilm.

Characterization of Aquatic Biofilms with Flow Cytometry

The precise identification of fibro-adipogenic progenitor cells (FAPs) and muscle stem cells (MuSCs) is critical to studying their biological function in physiological and pathological conditions. This protocol provides guidelines for the isolation, purification, and culture of FAPs and MuSCs from adult mouse muscles.

FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle

This protocol describes the use of fluorescence-activated cell sorting of human mesenchymal stem cells using the single-cell sorting method. Specifically, the use of single-cell sorting can achieve 99% purity of the immunophenotyped cells from a heterogeneous population when combined with a multiparametric flow cytometry-based approach.

Single-Cell Sorting of Immunophenotyped Mesenchymal Stem Cells from Human Exfoliated Deciduous Teeth

This protocol provides a standardized method for isolating and enriching primary myoblasts from adult and neonatal mouse skeletal muscle through tissue dissociation, sequential filtration, preplating, and defined culture conditions, enabling the establishment and maintenance of satellite cell-derived myoblast cultures for downstream experimental applications.

Isolation of Skeletal Muscle Satellite Cells for In Vitro Myogenesis Studies

Learn fluorescence-activated cell sorting to isolate live scleractinian coral cell populations with high specificity. This protocol enables functional assays and transcriptomic studies for marine biology research.

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

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