We thank Dr. Yitzchak Oschry and Dr. Orit Sagi-Asif for their help with FACS sorting. This research was supported by grants to NE from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n &deg; [276890], from the Israel Cancer Association (#20110078), and from the Israel Cancer Research Fund (Research Career Development Award).

1. Dissecting Mammary or Skin Tissue from Mice
<ol>
	<li>Before dissecting the desired tissue from mice, prepare the following supplies and reagents:
	 Supplies: 
	<ul>
		<li>Surgery tools (washed with detergent and then dipped in 70% ethanol).</li>
		<li>Styrofoam surface to rest the mouse on during dissection.</li>
		<li>Pins or needles to hold mice during dissection.</li>
		<li>Glass jar with lid for digestion, containing magnetic stir bar (autoclaved).</li>
		<li>Water bath at 37 &deg;C, containing a submersible magnetic stirrer and enough water to submerge the digestion jar, while resting on the stir plate.</li>
	</ul>
	 Reagents:
	<ul>
		<li>FACS buffer I: (4 &deg;C): Dulbecco's Phosphate Buffered Saline CMF (Calcium Magnesium Free) + 0.5% BSA</li>
		<li>Collagenase solution (made immediately before use): 
		0.05 g Collagenase II 
		0.05 g Collagenase IV 
		0.01 g Deoxyribonuclease 
		20 ml FACS buffer I. Keep collagenase solution on ice until use.</li>
		<li>PharmLyse (Ammonium Chloride Lysing Reagent).</li>
		<li>FACS buffer II: Dulbecco's Phosphate Buffered Saline CMF + 1% FCS.</li>
	</ul>
	</li>
	<li>Dissection of mammary glands (see also17).
	<ul>
		<li>Euthanize female mice using carbon dioxide (CO2) inhalation.</li>
		<li>On a Styrofoam surface, place the mouse on its back and pin firmly all four limbs using 25 gauge needles (Figure 1A). Spray the mouse generously with 70% ethanol.</li>
		<li>Using forceps, pull up the abdominal skin at the midline and make a small incision with sharp scissors.</li>
		<li>Starting from the incision, cut the skin up to the neck of the animal while avoiding puncturing the abdominal or thoracic cavities.</li>
		<li>Cut the skin on the rear legs from the midline incision towards the leg, resulting in a "Y shape".</li>
		<li>Pull skin away from mouse body and pin down, exposing the mammary glands attached to the underside of the skin (Figure 1B).</li>
		<li>Use Q-tips to gently separate mammary glands from skin while cutting conjunctive tissue with small sharp scissors to separate the mammary gland towards the spine of the mouse.</li>
		<li>The abdominal glands (#4, 5) are best for this protocol. You can also use the 2nd mammary gland, but bear in mind that these glands are anatomically located in close proximity to the pectoralis muscle which may be hard to separate, resulting in cells of mixed tissue origin.</li>
		<li>Note: When dissecting tumor tissue, it is recommended to remove necrotic regions.</li>
	</ul>
	</li>
	<li>Dissection of skin tissue: The easiest way to obtain skin tissue, without having to deal with hair removal is to use mouse ears. If a larger amount of tissue is required, you can shave off hair from mouse torso and dissect skin.
	<ul>
		<li>Euthanize mice using CO2 inhalation.</li>
		<li>Using sharp scissors, cut off both ears and place immediately in digestion jar containing a small volume (~0.5 ml) of PBS on ice.</li>
	</ul>
	</li>
</ol>
2. Preparation of Mammary Gland/skin Single Cell Suspension
<ul>
	<li>Place mammary glands/skin in digestion jar containing a small volume of PBS on ice. If tissue is bloody wash x2 with PBS, and then discard excess PBS.</li>
	<li>Mince thoroughly with curved scissors.</li>
	<li>Add 20 ml collagenase solution (Note: this amount of collagenase solution will efficiently dissociate approximately 5 g of mammary or tumor tissue and ears from up to 15 mice).</li>
	<li>Place immediately on stir plate in 37 &deg;C water bath, and incubate for 15 min while stirring at medium rate.</li>
</ul>
Note: optimal incubation times may vary if digesting other tissues.
<ul>
	<li>To stop the reaction, add 30 ml cold DMEM + 10% FCS.</li>
	<li>Strain the tissue/media mixture through a 70 &mu;m cell strainer placed on top of a 50 ml conical tube.</li>
	<li>Centrifuge the conical tube for 5 min at 450 x g at 4 &deg;C.</li>
</ul>
3. Red Blood Cells Lysis
Note: this step is not necessary if mice were heart-perfused with PBS before they were sacrificed.
<ul>
	<li>Aspirate the supernatant using a glass pipette attached to vacuum, without disturbing the cell pellet.</li>
	<li>To lyse RBC, resuspend cell pellet in 10 ml PharmLyse solution and incubate for 5 min at room temperature.</li>
	<li>Neutralize the PharmLyse solution by adding approximately 40 ml of FACS buffer I.</li>
	<li>Spin the conical tube with cells for 5 min at 450 x g at 4 &deg;C.</li>
	<li>Aspirate the supernatant.</li>
</ul>
4. Blocking of Endogenous Fc
<ul>
	<li>Optional: count cells to determine the appropriate volume for the following steps.</li>
	<li>Resuspend cells in 1-3 ml FACS buffer I (depending on cell number, and amount of antibody you want to use).</li>
	<li>Add FcBlock (anti-mouse CD16/CD32) to reach 1:50 dilution (10 &mu;g/ml), and incubate on ice for 10-20'. 
	No need to wash off FcBlock.</li>
</ul>
5. Staining
<ul>
	<li>Take out 100 &mu;l aliquots to eppendorf tubes to be used as an un-stained control and single color controls (according to the number of fluorescent antibodies used).</li>
	<li>To label fibroblasts: add anti-PDGFR&alpha; (directly conjugated to fluorophore) to a dilution of 1:50 (10 &mu;g/ml).</li>
	<li>To label other cell populations (e.g. immune cells, macrophages, endothelial cells etc.): add appropriate fluorescently conjugated antibodies to surface markers, to a 1:50 dilution.</li>
</ul>
Note: although PDGFR&alpha; is a robust marker for fibroblasts, it is recommended to include antibodies for immune cells and for epithelial cells in order to exclude any double positive populations and thus enhance the purity of isolated fibroblasts.
<ul>
	<li>Add 2 &mu;l of each antibody to each of the single color control eppendorf tubes.</li>
	<li>Incubate cells with antibodies for 1 hr on ice in the dark.</li>
	<li>To wash: fill tubes with FACS buffer I and spin for 5 min at 450 x g at 4 &deg;C.</li>
	<li>Aspirate supernatant, resuspend cells in FACS buffer I to a concentration of approximately 2 x 106 cells/ml, or whichever concentration most appropriate for sorting with your available FACS sorter.</li>
</ul>
Optional: Cells may be resuspended in FACS buffer II for the duration of sorting. This may improve cell viability. However, exposure to factors in serum may have an effect gene expression, which should be tested, and taken into account.
<ul>
	<li>Transfer labeled cells into FACS tubes with filter top: filter cells into the tubes to prevent cell clumps.</li>
	<li>To exclude dead cells, add 7AAD (0.5 &mu;g/ml) or Propidium Iodide (PI) to samples (except for the un-stained control).</li>
</ul>
Note: To avoid wasting cells, you can add 7AAD/PI to the un-stained sample after recording in the FACS, and use again as 7AAD-only control.
<ul>
	<li>Keep samples on ice while analyzing.</li>
</ul>
6. Isolation of Cells by FACS
(Note: this part of the protocol requires previous knowledge in operating a FACS sorter, e.g. BD FACS AriaII, or the assistance of a skilled technician).
<ul>
	<li>Using the FACS sorter software (BD FACSDiva), prepare appropriate analysis plots to analyze forward scatter (FSC), side scatter (SSC), 7AAD, FITC, PE and any other fluorophore used to label cells (Figure 2).</li>
	<li>Before loading each sample to the cell sorter, vortex briefly to resuspend cells.</li>
	<li>Start by analyzing the unstained sample in order to set the gating for the total cell population, to gate out cell debris, and to determine autofluorescence or background staining.</li>
	<li>Analyze unstained sample + 7AAD to determine and gate the population of live cells from the total cell population.</li>
	<li>Analyze each single color control to determine and calibrate parameters such as compensation.</li>
	<li>Analyze the stained sample to define the position of gates for sorting.</li>
	<li>Once parameters and gates for the desired cell populations have been set, begin sorting the labeled cell populations into eppendorf tubes or 15 ml conical tubes containing culture medium or RNA lysis buffer (such as RLT buffer or Trizol).</li>
</ul>
Note: If sorting a large number of cells, it is not recommended to sort directly into RNA lysis buffer, as the large volume of sheath fluid accompanying the cells will dilute the lysis buffer and reduce the yield.
<ul>
	<li>Spin cells down immediately after sorting is finished, and transfer into fresh medium or RNA lysis buffer, depending on the purpose of your experiment.</li>
	<li>If sorting in order to culture isolated cells, perform sterile sort. For better recovery of cells, use phenol red free DMEM + 5% FCS instead of FACS buffer II and collect cells into culture medium with 20% FCS.</li>
	<li>If culturing sorted fibroblasts, alwaysplate on Collagen coated plates and never directly on plastic as this results in activation of fibroblasts leading to changes in their gene expression.</li>
</ul>

<ol>
	<li>Kalluri, R., Zeisberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+in+cancer.">Fibroblasts in cancer.</a> Nat. Rev. Cancer. 6, 392-401 (2006).</li><li>Shimoda, M., Mellody, K. T., Orimo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carcinoma-associated+fibroblasts+are+a+rate-limiting+determinant+for+tumour+progression.">Carcinoma-associated fibroblasts are a rate-limiting determinant for tumour progression.</a> Seminars in cell &amp; developmental biology. 21, 19-25 (2010).</li><li>Orimo, A., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+stromal+fibroblasts+in+tumors.">Heterogeneity of stromal fibroblasts in tumors.</a> Cancer Biol. Ther. 6, 618-619 (2007).</li><li>Ostman, A., Augsten, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-associated+fibroblasts+and+tumor+growth--bystanders+turning+into+key+players.">Cancer-associated fibroblasts and tumor growth--bystanders turning into key players.</a> Current opinion in genetics &amp; development. 19, 67-73 (2009).</li><li>Allinen, 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=Molecular+characterization+of+the+tumor+microenvironment+in+breast+cancer.">Molecular characterization of the tumor microenvironment in breast cancer.</a> Cancer Cell. 6, 17-32 (2004).</li><li>Bhowmick, N. A., Neilson, E. G., Moses, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+fibroblasts+in+cancer+initiation+and+progression.">Stromal fibroblasts in cancer initiation and progression.</a> Nature. 432, 332-337 (2004).</li><li>Orimo, A., 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=Stromal+fibroblasts+present+in+invasive+human+breast+carcinomas+promote+tumor+growth+and+angiogenesis+through+elevated+SDF-1/CXCL12+secretion.">Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion.</a> Cell. 121, 335-348 (2005).</li><li>Pietras, K., Ostman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+interactions+with+the+tumor+stroma.">Hallmarks of cancer: interactions with the tumor stroma.</a> Experimental cell research. 316, 1324-1331 (2010).</li><li>Erez, N., Truitt, M., Olson, P., Arron, S. T., Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-Associated+Fibroblasts+Are+Activated+in+Incipient+Neoplasia+to+Orchestrate+Tumor-Promoting+Inflammation+in+an+NF-kappaB-Dependent+Manner.">Cancer-Associated Fibroblasts Are Activated in Incipient Neoplasia to Orchestrate Tumor-Promoting Inflammation in an NF-kappaB-Dependent Manner.</a> Cancer Cell. 17, 135-147 (2010).</li><li>Sugimoto, H., Mundel, T. M., Kieran, M. W., Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+fibroblast+heterogeneity+in+the+tumor+microenvironment.">Identification of fibroblast heterogeneity in the tumor microenvironment.</a> Cancer Biol. 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G., 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=Leukocyte+complexity+in+breast+cancer+predicts+overall+survival+and+functionally+regulates+response+to+chemotherapy.">Leukocyte complexity in breast cancer predicts overall survival and functionally regulates response to chemotherapy.</a> Cancer Discovery. 1, 54-67 (2011).</li><li>Pahler, J. C., 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=Plasticity+in+tumor-promoting+inflammation:+impairment+of+macrophage+recruitment+evokes+a+compensatory+neutrophil+response.">Plasticity in tumor-promoting inflammation: impairment of macrophage recruitment evokes a compensatory neutrophil response.</a> Neoplasia. 10, 329-340 (2008).</li><li>Pietras, K., Pahler, J., Bergers, G., Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+paracrine+PDGF+signaling+in+the+proangiogenic+tumor+stroma+revealed+by+pharmacological+targeting.">Functions of paracrine PDGF signaling in the proangiogenic tumor stroma revealed by pharmacological targeting.</a> PLoS Med. 5, e19 (2008).</li><li>Quante, 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=Bone+marrow-derived+myofibroblasts+contribute+to+the+mesenchymal+stem+cell+niche+and+promote+tumor+growth.">Bone marrow-derived myofibroblasts contribute to the mesenchymal stem cell niche and promote tumor growth.</a> Cancer Cell. 19, 257-272 (2011).</li><li>Plante, I., Stewart, M. 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No conflicts of interest declared.

While experiments performed in tissue culture can be informative and suggest functional principles that can be verified in vivo, it is known that large changes occur in gene expression of cells in culture11,12 . In order to avoid a tissue culture step when profiling gene expression in fibroblasts, we developed a protocol that allows isolation of normal, as well as cancer-associated fibroblasts from fresh mouse or human tissue. This protocol was successfully applied with skin, pancreas and breast tissu...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Gibco</td>
 <td>41965</td>
 <td></td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Biological Industries</td>
 <td>02-023-1A</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase II</td>
 <td>Worthington</td>
 <td>LS4176</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase IV</td>
 <td>Worthington</td>
 <td>LS4188</td>
 <td></td>
 </tr>
 <tr>
 <td>Deoxyribonuclease </td>
 <td>Worthington</td>
 <td>LS2007</td>
 <td></td>
 </tr>
 <tr>
 <td>PharmLyse</td>
 <td>BD</td>
 <td>555899</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell strainer 70 &mu;m</td>
 <td>SPL</td>
 <td>93070</td>
 <td></td>
 </tr>
 <tr>
 <td>Purified anti-mouse CD16/CD32</td>
 <td>BD Pharmingen</td>
 <td>553142</td>
 <td></td>
 </tr>
 <tr>
 <td>Via probe (7AAD)</td>
 <td>e-Bioscience</td>
 <td>00-6993-50</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse CD140a-PE (PDGFRa)</td>
 <td>e-Bioscience</td>
 <td>12-1401-81</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse F4/80- FITC</td>
 <td>Cederlane</td>
 <td>CL8940F</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM w/o Phenol Red</td>
 <td>Gibco</td>
 <td>31053</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagen Type I</td>
 <td>BD Biosciences</td>
 <td>354236</td>
 <td></td>
 </tr>
 </tbody>
 </table>

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.

Using PDGFR&alpha; as a marker for fibroblasts results in isolation of highly enriched populations of tissue fibroblasts. The level of purity after sorting was 99%, as quantified by post-sort analysis (Figure 2A). Estimating the percentage of contaminating non-fibroblast cells by the relative expression of cell-specific control genes (Figure 2B) typically shows 0.1-0.6% contamination. This level of purity allows high quality transcriptome profiling of isolated fibroblasts9.</p...

Watch this Scientific Journal Video about Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS at JoVE.com

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.

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 ...

isolation-normal-cancer-associated-fibroblasts-from-fresh-tissues

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35.6K vues.  Tel Aviv University. Cancer fibroblastes associés (CAF) de faciliter l'initiation des tumeurs, la croissance et la progression à travers signalisation qui favorise la prolifération, l'angiogenèse et l'inflammation. Nous décrivons ici une méthode pour isoler des populations pures de fibroblastes normaux et de souris CAF frais et tissus humains par tri cellulaire, en utilisant PDGFRα comme un marqueur de surface.

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

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

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 ...

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

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 ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

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 ...

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 ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...