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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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

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 ...

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Research

JoVE Journal

Biology

7.9K Views.  University of Miami. 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.

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

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

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.

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Developmental Biology

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

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

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.

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

Characterization of Aquatic Biofilms with Flow Cytometry

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

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.

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond ...

Environment

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 ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Bioengineering

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

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

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 (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Immunology and Infection

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

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

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.

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Medicine

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

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along fibrogenic, adipogenic, osteogenic, or chondrogenic lineage. Together with muscle stem cells (MuSCs), FAPs play a critical role in muscle homeostasis, repair, and regeneration, while actively maintaining and remodeling the extracellular matrix (ECM). In pathological conditions, such as chronic damage and muscular dystrophies, FAPs undergo aberrant activation and differentiate into collagen-producing fibroblasts and adipocytes, leading to fibrosis and intramuscular fatty infiltration. Thus, FAPs play a dual role in muscle regeneration, either by sustaining MuSC turnover and promoting tissue repair or contributing to fibrotic scar formation and ectopic fat infiltrates, which compromise the integrity and function of the skeletal muscle tissue. A proper purification of FAPs and MuSCs is a prerequisite for understanding the biological role of these cells in physiological as well as in pathological conditions. Here, we describe a standardized method for the simultaneous isolation of FAPs and MuSCs from limb muscles of adult mice using fluorescence-activated cell sorting (FACS). The protocol describes in detail the mechanical and enzymatic dissociation of mononucleated cells from whole limb muscles and injured tibialis anterior (TA) muscles. FAPs and MuSCs are subsequently isolated using a semi-automated cell sorter to obtain pure cell populations. We additionally describe an optimized method for culturing quiescent and activated FAPs and MuSCs, either alone or in coculture conditions.

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.

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along ...

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 ...

Multicellular Spheroid Formation for FLIM-Based Metabolic and Hypoxia Analysis

Production and Multi-Parameter Live Cell Fluorescence Lifetime Imaging Microscopy (FLIM) of Multicellular Spheroids

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug therapies&#160;and using bio- and nanosensors in a 3D context. Their ease of production, predictable size, growth, and observed nutrient and metabolite gradients are important to recapitulate the 3D niche-like&#160;cell microenvironment. However, spheroid heterogeneity and variability of their production methods can influence overall cell metabolism, viability, and drug response. This makes it difficult to choose the most appropriate&#160;methodology, considering the requirements in size, variability, needs of biofabrication, and use as in vitro 3D tissue models in stem and cancer cell biology. In particular, spheroid production can influence their compatibility with quantitative live microscopies, such as optical metabolic imaging, fluorescence lifetime imaging microscopy (FLIM), monitoring of spheroid&#160;hypoxia with nanosensors, or viability. Here, a number of conventional spheroid formation protocols are presented, highlighting their compatibility with the live widefield, confocal, and two-photon microscopies. The follow-up imaging to analysis&#160;pipeline with multiplexed autofluorescence FLIM and, using various types of cancer and stem cell spheroids, is also presented.

Author Spotlight: Standardizing Spheroid Formation Methods for Metabolic and Oxygenation Analysis Using Fluorescence Lifetime Imaging Microscopy

Here, we describe different multicellular spheroid formation methods to perform follow-up multi-parameter live cell microscopy. Using fluorescence lifetime imaging microscopy (FLIM), cellular autofluorescence, staining dyes, and nanoparticles, the approach for analysis of cell metabolism, hypoxia, and cell death in live three-dimensional (3D) cancer and stem cell-derived spheroids is demonstrated.

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug ...