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>
	<li>Bellwood, D. R., Hughes, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional-scale+assembly+rules+and+biodiversity+of+coral+reefs.">Regional-scale assembly rules and biodiversity of coral reefs.</a> Science. 292 (5521), 1532-1535 (2001).</li><li>Ferrario, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effectiveness+of+coral+reefs+for+coastal+hazard+risk+reduction+and+adaptation.">The effectiveness of coral reefs for coastal hazard risk reduction and adaptation.</a> Nature Communications. 5 (3794), 1-9 (2014).</li><li>Wegley, L., Edwards, R., Rodriguez-Brito, B., Liu, H., Rohwer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenomic+analysis+of+the+microbial+community+associated+with+the+coral+Porites+astreoides.">Metagenomic analysis of the microbial community associated with the coral Porites astreoides.</a> Environmental Microbiology. 9 (11), 2707-2719 (2007).</li><li>Gates, R. 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Alternative interpretations of the experimental and ecological data.</a> Journal of Experimental Marine Biology and Ecology. 346 (1), 36-44 (2007).</li><li>Miller, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coral+disease+following+massive+bleaching+in+2005+causes+60%+decline+in+coral+cover+on+reefs+in+the+US+Virgin+Islands.">Coral disease following massive bleaching in 2005 causes 60% decline in coral cover on reefs in the US Virgin Islands.</a> Coral Reefs. 28 (4), 925-937 (2009).</li><li>Hoegh-Guldberg, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coral+reefs+under+rapid+climate+change+and+ocean+acidification.">Coral reefs under rapid climate change and ocean acidification.</a> Science. 318 (5857), 1737-1742 (2007).</li><li>Berkelmans, R., Van Oppen, M. J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+zooxanthellae+in+the+thermal+tolerance+of+corals:+A+"nugget+of+hope"+for+coral+reefs+in+an+era+of+climate+change.">The role of zooxanthellae in the thermal tolerance of corals: A "nugget of hope" for coral reefs in an era of climate change.</a> Proceedings of the Royal Society B: Biological Sciences. 273 (1599), 2305-2312 (2006).</li><li>Cunning, R., Silverstein, R. N., Baker, A. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy.">Anatomy.</a> Diseases of Coral. , 85-107 (2015).</li><li>Allemand, D., Tambutt&#233;, &. #. 2. 0. 1. ;., Zoccola, D., Tambutt&#233;, S., Dubinsky, Z., Stambler, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coral+calcification,+cells+to+reefs.">Coral calcification, cells to reefs.</a> Coral Reefs: An Ecosystem in Transition. , 119-150 (2011).</li><li>Rinkevich, B., Loya, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Reproduction+of+the+Red+Sea+Coral+Stylophora+pistillata.+I.+Gonads+and+Planulae.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corals+use+similar+immune+cells+and+wound-healing+processes+as+those+of+higher+organisms.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+observations+of+recovery+in+the+reef-building+scleractinian+coral,+Montastrea+annularis,+after+bleaching+on+a+Cayman+reef.">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., Muscatine, L., Lenhoff, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cnidarian+Histology.">Cnidarian Histology.</a> Coelenterate Biology. , 1-43 (1974).</li><li>Traylor-Knowles, N., Rose, N. H., Palumbi, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+specificity+of+gene+expression+in+the+response+to+heat+stress+in+corals.">The cell specificity of gene expression in the response to heat stress in corals.</a> The Journal of Experimental Biology. 220 (10), 1837-1845 (2017).</li><li>Traylor-Knowles, N., Palumbi, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+environmental+biology:+Cell+biology+informing+conservation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coral+cell+separation+and+isolation+by+fluorescence-activated+cell+sorting+(FACS).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+mammalian-like+haematopoietic+system+found+in+a+colonial+chordate.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+Symbiodinium+Strains+Are+Hosted+by+the+Brazilian+Endemic+Corals+Mussismilia+spp.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+enumeration+of+bacterial+in+the+coral+surface+mucus+layer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bleaching+response+of+Symbiodinium+(zooxanthellae):+Determination+by+flow+cytometry.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genome+of+Aiptasia,+a+sea+anemone+model+for+coral+symbiosis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+the+genomes+of+Stylophora+pistillata+and+Acropora+digitifera+provides+evidence+for+extensive+differences+between+species+of+corals.">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><li>Pavlov, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydraulic+control+of+tuna+fins:+A+role+for+the+lymphatic+system+in+vertebrate+locomotion.">Hydraulic control of tuna fins: A role for the lymphatic system in vertebrate locomotion.</a> Science. 357 (6348), 310-314 (2017).</li></ol>

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.

fluorescence-activated-cell-sorting-for-isolation-scleractinian-cell

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Biology

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Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

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.

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification of bacterial taxa that transform specific pools of DOC in nature poses a technical challenge. 
Here we describe an approach that couples bromodeoxyuridine (BrdU) incorporation, fluorescence activated cell sorting (FACS), and 16S rRNA gene-based molecular analysis that allows culture-independent identification of bacterioplankton capable of degrading a specific DOC compound in aquatic environments. Triplicate bacterioplankton microcosms are set up to receive both BrdU and a model DOC compound (DOC amendments), or only BrdU (no-addition control). BrdU substitutes the positions of thymidine in newly synthesized bacterial DNA and BrdU-labeled DNA can be readily immunodetected 1,2. Through a 24-hr incubation, bacterioplankton that are able to use the added DOC compound are expected to be selectively activated, and therefore have higher levels of BrdU incorporation (HI cells) than non-responsive cells in the DOC amendments and cells in no-addition controls (low BrdU incorporation cells, LI cells). After fluorescence immunodetection, HI cells are distinguished and physically separated from the LI cells by fluorescence activated cell sorting (FACS) 3. Sorted DOC-responsive cells (HI cells) are extracted for DNA and taxonomically identified through subsequent 16S rRNA gene-based analyses including PCR, clone library construction and sequencing.

Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Environmental bacterioplankton are incubated with a model dissolved organic carbon (DOC) compound and a DNA labeling reagent, bromodeoxyuridine (BrdU). Afterward, DOC-degrading cells are separated from the bulk community based on their elevated BrdU incorporation using fluorescence activated cell sorting (FACS). These cells are then identified by subsequent molecular analyses.

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification ...

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

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

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

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

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 PDGFR&#x03B1; as a surface marker.

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...