The authors would like to thank both Jean-Pierre Baudoin and Olivier Mbarek for their advice and Claire Andr&#233;ani for her help in English corrections and modifications. This work was supported by a grant from the French State managed by the National Research Agency under the &#34;Investissements d&#8217;avenir (Investments for the Future)&#34; program with the reference ANR-10-IAHU-03 (M&#233;diterran&#233;e Infection) and by R&#233;gion Provence Alpes C&#244;te d&#8217;Azur and European funding FEDER PRIMI.

1. Amoeba Culture
<ol>
	<li>Use Vermamoeba vermiformis (strain CDC19) as a cell support.</li>
	<li>Add 30 mL of protease-peptone-yeast extract-glucose medium (PYG) (Table 1) and 3 mL of the amoebae at a concentration of 1 x 106 cells/mL in a 75 cm2 cell culture flask.</li>
	<li>Maintain the culture at 28 &#176;C.</li>
	<li>After 48 h, quantify the amoebae using counting slides.</li>
	<li>To rinse, harvest the cells at a concentration of 1 x 106 cells/mL and pellet the amoebae by centrifugation at 720 x g for 10 min. Remove the supernatant and resuspend the pellet in the appropriate volume of starvation medium to obtain 1 x 106 cells/mL (Table 1).</li>
</ol>
2. Propagation of Stock Virus in Amoebae
NOTE: Before dilution, it is important to culture the stock sample to obtain enough fresh culture, then proceed to filtration.
<ol>
	<li>Use 1 x 106 of amoeba culture in starvation medium.</li>
	<li>Inoculate the mixture of viruses issued from the stock solution (Faustovirus/Usurpativirus LCD7 or Faustovirus/Clandestinovirus ST1) after the co-culture process on the cell support at a multiplicity of infection (MOI)&#160;of 0.01. 
	NOTE: The MOI is important to reduce the abundance of the major viral population and the number of infected cells.</li>
	<li>Incubate at 30 &#176;C until cytopathic effects (CPE) are induced, such as amoebal rounding or lysis, approximately 10 to 14 h after infection.</li>
	<li>Collect the media and filtrate through a 5 &#181;m filter to remove cellular debris.</li>
</ol>
3. End-point dilution
<ol>
	<li>Perform a serial dilution (10-1 to 10-11) of the viral sample in starvation medium (Table 1).</li>
	<li>Inoculate 2 mL of 1 x 106 Vermamoeba vermiformis contained in each Petri dish with 100 &#181;L of the mixture inoculum.</li>
	<li>Place the Petri dishes into a sealable plastic bag at 30 &#176;C.</li>
	<li>Begin observing the Petri dishes with inverted optical microscopy at 6 h postinfection and check cell morphology every 4 to 8 h.</li>
	<li>At the appearance of the cytopathic effect characterized by rounding cells, begin the single cell micro-aspiration process.</li>
</ol>
4. Single Cell Micro-aspiration
<ol>
	<li>Prepare the host. 
	NOTE: This preparation is made for the release of infected single cells to a fresh cell support.
	<ol>
		<li>Treat the amoebae in the culture with an antimicrobial agent containing 10 &#181;g/mL of vancomycin, 10 &#181;g/mL of imipenem, 20 &#181;g/mL of ciprofloxacin, 20 &#181;g/mL of doxycycline, and 20 &#181;g/mL of voriconazole. This mixture is used to avoid bacterial and fungal contamination. 
		NOTE: The procedure takes place on a bench outside the microbiological safety station. Add 2 mL of amoebae concentrated at 1 x 106 cells/mL each into 15 Petri dishes. For amoeba adherence, incubate the culture at 30 &#176;C for 30 min.</li>
	</ol>
	</li>
	<li>Select the Petri dish used for the micro-aspiration from the limit dilution according to the following criteria: 1) absence of any visible contamination by fungal and bacterial agents, 2) evidence of cytopathic effect of amoebae due to the viruses, and 3) prelysis and rounding phase of the amoebae (to avoid aspiration of viral particles).</li>
	<li>Set up a workstation with the following materials (see Figure 1A,B): 
	Micromanipulator, which allows microcapillary positioning; 
	Manual control pressure device, used to aspirate and release the cells into the microcapillary; 
	Inverted microscope; 
	Plug and play motor modules; 
	Camera; 
	Computer module to visualize manipulation and take pictures.</li>
	<li>Choose a microcapillary (see Figure 1C). 
	NOTE: The size of the cells, the deformation and adhesion of their membranes to the surfaces, and the cellular motility can impact the smooth progress of the micro-aspiration. The microcapillary diameter can be precisely chosen and adapted to specific cell types depending on their sizes and methods of aspiration. A microcapillary of 20 &#181;m inner diameter was used to aspirate a rounding amoeba (diameter ~10 &#181;m). This allows the upkeep of an internal position and an easy release of the cell.</li>
	<li>Mount the system.
	<ol>
		<li>Fix the operating angle of the gripping system on the motorized module at 45&#176;.</li>
		<li>Perform a double installation, first on the gripping system, and then on the microcapillary.</li>
		<li>Focus on the cells after running a few drops of oil through the microcapillary. 
		NOTE: The mineral oil with biological compatibility is supplied by the device.</li>
		<li>Complete mounting following manufacturer&#39;s recommendations.</li>
	</ol>
	</li>
	<li>Clone cells (see Figure 2A,B). 
	NOTE: This procedure is similar to the one described by Fr&#246;hlich and K&#246;nig31.
	<ol>
		<li>Place the Petri dish containing 2 mL of infected amoebae under the microscope.</li>
		<li>Focus first on the cells, and then on the microcapillary immersed in the culture.</li>
		<li>Pick a rounded single cell and bring the microcapillary closer to the micromanipulator.</li>
		<li>Exert soft aspiration with manual pressure control on the cell, taking it inside the microcapillary. Remove the single cell from the first sample and release in the cellular support, then incubate it at 30 &#176;C.</li>
		<li>Conduct daily observations with an inverted optical microscope to observe the appearance of the cells and to monitor the emergence of the cytopathic effect.</li>
	</ol>
	</li>
</ol>
5. PCR Screening
NOTE: Following step 4, a systematic screening by PCR is crucial to confirm the separation. In both Usurpativirus/Faustovirus and Clandestinovirus/Faustovirus, the design and application of the specific primer and probe systems were done using Primer-BLAST online32 (Table 2).
<ol>
	<li>Extract DNA from a part of the positive culture samples (i.e., where a cytopathic effect is observed), using an automated extraction system according to the manufacturer&#39;s protocol.</li>
	<li>Use appropriately designed primers. 
	NOTE: Here we designed primers to amplify core genes annotated as RpB2 (Faustovirus), LCD7 major capsid protein (Usurpatvirus) and minor capsid protein (Clandestinovirus)</li>
	<li>Perform standard PCR using a thermocycler.
	<ol>
		<li>Carry out 20 &#181;L PCR reactions with 50 &#181;M of each primer (Table 2), 1x Master Mix, and RNase free water.</li>
		<li>Activate the Taq DNA polymerase for 5 min at 95 &#176;C, then follow with 45 cycles of 10 s denaturation at 95 &#176;C, annealing of the primers for 30 s at 58 &#176;C, and extension for 30 s at 72 &#176;C.</li>
	</ol>
	</li>
	<li>Run the PCR products on a 1.5% agarose gel, stain with DNA gel stain (Table of Materials), and visualize with UV.</li>
</ol>
6. Virus Production and Purification
<ol>
	<li>Put the rest of the Petri dish culture back in a small flask.</li>
	<li>For the virus production, prepare 15 flasks of 145 cm2, containing 40 mL of Vermamoeba vermiformis in starvation medium and 5 mL of the isolated virus already transferred from the Petri dish to small flasks.</li>
	<li>Treat with the same antibiotic and antifungal mixture used in step 4.1.</li>
	<li>Incubate at 30 &#176;C. Observe every day with inverted optical microscopy.</li>
	<li>After the complete infection, pool all flasks. Use a 0.45 &#181;m filter to eliminate debris.</li>
	<li>Ultracentrifuge all supernatants at&#160;50,000 x g for&#160;45 min.</li>
	<li>After centrifugation, remove the supernatant from each tube by aspiration and resuspend the pellet in 1 mL of phosphate buffered saline (PBS).</li>
	<li>Purify the virus produced using 25% sucrose (27.5 g sucrose in 100 mL of PBS, sterilized by filtration).</li>
	<li>Centrifuge 8 mL of sucrose and 2 mL of the viral suspension at&#160;80,000 x g for&#160;30 min. Resuspend the viral pellet in 1 mL of PBS. Store it at -80 &#176;C.</li>
</ol>
7. Negative Staining and Transmission Electron Microscopy
NOTE: Bou Khalil et al. previously published this protocol27.
<ol>
	<li>Deposit 5 &#956;L of the lysis supernatant onto the glow-discharged grid. Leave for approximately 20 min at room temperature. 
	NOTE: The glow-discharge allows us to obtain a hydrophilic grid by plasma application.</li>
	<li>Dry the grid carefully and deposit a small drop of 1% ammonium molybdate on it for 10 s. Leave the grid to dry for 5 min.</li>
	<li>Proceed to electron microscopy observations at 200 keV.</li>
</ol>
8. Characterization of Clandestinovirus ST1 and Usurpativirus LCD7
<ol>
	<li>Characterize pure populations of Clandestinovirus ST1 and Usurpativirus LCD7 using genome sequencing, genome assembly, bioinformatics analyses, and study of their replicative cycle as we have done for other viruses10,20,29.</li>
</ol>

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All authors have nothing to disclose.

The duration of the single cell micro-aspiration handling and its good functioning is operator-dependent. The different steps of the experiment require precision. The use of the micromanipulation components of the workstation must be under constant control by observing the process of micro-aspiration and the release of the cell. The follow-up by microscopic observation is necessary for capture and transfer of a cell. An experienced operator can take 1 to 2 h to isolate 10 cells and retransfer them one by one depending on...

Nucleocytoplasmic large DNA viruses (NCLDV) are extremely diverse, defined by four families that infect eukaryotes1. The first described viruses with genomes above 300 kbp were Phydcodnaviridae, including Paramecium bursaria Chlorella virus 1 PBCV12. The isolation and the first description of Mimivirus, showed that the size of viruses doubled in terms of both the size of the particle (450 nm) and the length of the genome (1.2 Mb)3. Since then, many giant viruses have been described, usually isolated using an amoeba co-culture procedure. Several giant viruses with different morphologies and genetic contents can be isolated from Acanthamoeba sp. cells, including Marseilleviruses, Pandoraviruses, Pithoviruses, Mollivirus, Cedratviruses, Pacmanvirus, Tupanvirus, and recently Medusavirus4,5,6,7,8,9,10,11,12,13,14,15,16,17. In parallel, the isolation of Vermamoeba vermiformis allowed the isolation and description of the giant viruses Faustovirus, Kaumoebavirus, and Orpheovirus18,19,20. Other giant viruses were isolated with their host protists, such as Cafeteria roenbergensis21, Aureococcus anophagefferens22, Chrysochromulina ericina23, and Bodo saltans24. All of these isolations were the result of an increasing number of teams working on isolation and the introduction of high throughput strategy updates25,26,27,28, such as the improvement of the co-culture system with the use of flow cytometry.
In 2016, we used a strategy associating co-culture and flow cytometry to isolate giant viruses27. This strategy was developed to increase the number of samples inoculated, to diversify protists used as cell supports, and to quickly detect the lysis of the cell support. The system was updated by adding a supplemental step to avoid preliminary molecular biology identification and quick detection of an unknown viral population as in the case of Pacmanvirus29. Coupling flow cytometry to cell sorting allowed for separation of a mixture of Mimivirus and Cedratvirus A1130. However, we later encountered the limitations of the separation and detection of these viral subpopulations by flow cytometry. After sequencing, when we assembled the genomes of Faustovirus ST125 and Faustovirus LCD7 (unpublished data), we surprisingly found in each assembly two supplemental genomes of two novel viruses not identified in public genome databases. However, neither flow cytometry nor transmission electronic microscopy (TEM) showed that the amoebaes were infected by two different viruses, Clandestinovirus ST1 and Usurpativirus LCD7. We designed specific PCR systems to amplify Faustovirus, Usurpativirus, and Clandestinovirus markers respectively based on their genomes; our purpose was to have PCR-based systems that enable verification of the purity of the viruses being separated. However, end-point dilution and flow cytometry failed to separate them. The isolation of this single viral population was difficult because neither the morphology nor replicative elements of Clandestinovirus and Usurpativirus populations have been characterized. We detected only one viral population by flow cytometry due to the overlapping of the two populations (tested after the effective separation). We tried to separate them using single particle sorting on 96-well plates, but we did not observe any cytopathic effects, and we detected neither Clandestinovirus nor Usurpativirus by PCR amplification. Finally, it was only the combination of end point dilution followed by single amoeba micro-aspiration that enabled separation of these two low-abundance giant viruses from Faustoviruses. This method of separation is the object of this article.

<table border="0" cellpadding="0" cellspacing="0" style="width:1068px;" width="1067">
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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Agarose Standard</td>
			<td style="width:185px;">Euromedex</td>
			<td style="width:161px;">Unkown</td>
			<td style="width:391px;">Standard PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">AmpliTaq Gold 360 Master Mix</td>
			<td style="width:185px;">Applied Biosystems</td>
			<td style="width:161px;">4398876</td>
			<td>Standard PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">CellTram 4r Oil</td>
			<td style="width:185px;">Eppendorf</td>
			<td style="width:161px;">5196000030</td>
			<td>Control the cells during the microaspiration process</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Corning cell culture flasks 150 cm2</td>
			<td style="width:185px;">Sigma-aldrich</td>
			<td style="width:161px;">CLS430825</td>
			<td>Culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Corning cell culture flasks 25 cm2</td>
			<td style="width:185px;">Sigma-aldrich</td>
			<td style="width:161px;">CLS430639</td>
			<td>Culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Corning cell culture flasks 75 cm2</td>
			<td style="width:185px;">Sigma-aldrich</td>
			<td style="width:161px;">CLS430641</td>
			<td>Culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">DFC 425C camera</td>
			<td style="width:185px;">LEICA</td>
			<td style="width:161px;">Unkown</td>
			<td>Observation/Monitoring</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Eclipse TE2000-S Inverted Microscope</td>
			<td style="width:185px;">Nikon</td>
			<td style="width:161px;">Unkown</td>
			<td>Observation/Monitoring</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">EZ1 advanced XL</td>
			<td style="width:185px;">Quiagen</td>
			<td style="width:161px;">9001874</td>
			<td>DNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Glasstic Slide 10 With Counting Grids</td>
			<td style="width:185px;">Kova International</td>
			<td style="width:161px;">87144E</td>
			<td>Cell count</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Mastercycler nexus</td>
			<td style="width:185px;">Eppendorf</td>
			<td style="width:161px;">6331000017</td>
			<td>Standard PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Microcapillary 20 &#181;m</td>
			<td style="width:185px;">Eppendorf</td>
			<td style="width:161px;">5175 107.004</td>
			<td>Microaspiration and release of cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Micromanipulator InjectMan NI2</td>
			<td style="width:185px;">Eppendorf</td>
			<td style="width:161px;">631-0210</td>
			<td>Microcapillary positioning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Nuclease-Free Water</td>
			<td style="width:185px;">ThermoFischer</td>
			<td style="width:161px;">AM9920</td>
			<td>Standard PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Optima XPN Ultracentrifuge</td>
			<td style="width:185px;">BECKMAN COULTER</td>
			<td style="width:161px;">A94469</td>
			<td>Virus purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Petri dish 35 mm</td>
			<td style="width:185px;">Ibidi</td>
			<td style="width:161px;">81158</td>
			<td>Culture/observation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Sterile syringe filters 5 &#181;m</td>
			<td style="width:185px;">Sigma-aldrich</td>
			<td style="width:161px;">SLSV025LS</td>
			<td>Filtration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">SYBR green Type I</td>
			<td style="width:185px;">Invitrogen</td>
			<td style="width:161px;">unknown</td>
			<td>Fluorescent molecular probes/ flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">SYBR Safe</td>
			<td style="width:185px;">Invitrogen</td>
			<td style="width:161px;">S33102</td>
			<td>Standard PCR; DNA gel stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Tecnai G20</td>
			<td style="width:185px;">FEI</td>
			<td style="width:161px;">Unkown</td>
			<td>Electron microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Type 70 Ti Fixed-Angle Titanium Rotor</td>
			<td style="width:185px;">BECKMAN COULTER</td>
			<td style="width:161px;">337922</td>
			<td>Virus purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Ultra-Clear Tube, 25 x 89 mm</td>
			<td style="width:185px;">BECKMAN COULTER</td>
			<td style="width:161px;">344058</td>
			<td>Virus purification</td>
		</tr>
	</tbody>
</table>

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.

Single cell micro-aspiration is a micromanipulation process optimized in this manuscript (Figure 1). This technique enables capture of a rounded, infected amoeba (Figure 2A) and its release in a novel plate containing uninfected amoebae (Figure 2). It is a functional prototype that applies to the co-culture system and has successfully isolated non-lytic giant viruses. This approach was used for the first time in the...

Watch this Scientific Journal Video about Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation at JoVE.com

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

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 ...

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7.0K Views.  Institut Hospitalo-Universitaire (IHU) - Méditerranée Infection. 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.

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

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 ...

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 ...

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 ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of pathways involved in parietal epithelial cell activation could therefore be a tool to attenuate the progression of glomerular diseases. This article describes a method to culture and analyze parietal epithelial cell outgrowth of encapsulated glomeruli isolated from mouse kidney. After dissecting isolated mouse kidneys, the tissue is minced, and glomeruli are isolated by sieving. Encapsulated glomeruli are collected, and single glomeruli are cultured for 6 days to obtain glomerular outgrowth of parietal epithelial cells. During this period, parietal epithelial cell proliferation and migration can be analyzed by determining the cell number or the surface area of outgrowing cells. This assay can therefore be used as a tool to study the effects of an altered gene expression in transgenic- or knockout-mice or the effects of culture conditions on parietal epithelial cell growth characteristics and signaling. Using this method, important pathways involved in the process of parietal epithelial cell activation and consequently in glomerulosclerosis can be studied.

This article describes a method for culturing and analyzing glomerular parietal epithelial cell outgrowths of encapsulated glomeruli isolated from mouse kidney. This method can be used to study pathways involved in parietal epithelial cell proliferation and migration.

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of ...

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.

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 ...

Real-Time Detection of Reactive Oxygen Species Production in Immune Response in Rice with a Chemiluminescence Assay

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen infection or challenge with pathogen-associated chemicals (pathogen-associated molecular patterns [PAMPs]), an array of immune responses, including a ROS burst, are quickly induced in plants, which is called PAMP-triggered immunity (PTI). A ROS burst is a hallmark PTI response, which is catalyzed by a group of plasma membrane-localized NADPH oxidases-the RBOH family proteins. The vast majority of ROS comprise hydrogen peroxide (H2O2), which can be easily and steadily detected by a luminol-based chemiluminescence method. Chemiluminescence is a photon-producing reaction in which luminol, or its derivative (such as L-012), undergoes a redox reaction with ROS under the action of a catalyst. This paper describes an optimized L-012-based chemiluminescence method to detect apoplast ROS production in real-time upon PAMP elicitation in rice tissues. The method is easy, steady, standardized, and highly reproducible under firmly controlled conditions.

Here, we describe a method for the real-time detection of apoplastic reactive oxygen species (ROS) production in rice tissues in pathogen-associated molecular pattern-triggered immune response. This method is simple, standardized, and generates highly reproducible results under controlled conditions.

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen ...