Wir danken Dr. Yitzchak Oschry und Dr. Orit Sagi-Asif für ihre Hilfe bei FACS-Sortierung. Diese Arbeit wurde durch Stipendien an NE von der Europäischen Union Siebten Rahmenprogramms (FP7/2007-2013) unter Finanzhilfevereinbarung n unterstützt ° [276890], aus dem Israel Cancer Association (# 20110078), und aus dem Israel Cancer Research Fund (Research Career Development Award).

Ein. Sezieren Mammary oder Skin Gewebe von Mäusen <ol><li> Vor der Zerlegung des gewünschten Gewebe von Mäusen, bereiten Sie die folgenden Lieferungen und Reagenzien: Supplies: <ul><li> Chirurgische Werkzeuge (gewaschen mit Reinigungsmittel getaucht und dann in 70% Ethanol). </li><li> Styropor Oberfläche mit der Maus auf beim Präparieren ruhen. </li><li> Pins oder Nadeln an Mäusen während der Präparation zu halten. </li><li> Glas mit Deckel für die Verdauung, mit Magnetrührstab (autoklaviert). </li><li> Wasserbad bei 37 ° C, mit einer Tauchpumpe Magnetrührer und genug Wasser zu tauchen die Verdauung jar, während es auf der Rührplatte. </li></ul> Reagenzien: <ul><li> FACS-Puffer I: (4 ° C): Dulbeccos Phosphat-gepufferte Saline CMF (Calcium Magnesium Gratis) + 0,5% BSA </li><li> Collagenase Lösung (aus unmittelbar vor der Verwendung): 0,05 g Collagenase II 0,05 g Kollagenase IV 0,01 g Deoxyribonucpachten 20 ml FACS-Puffer I. Halten Kollagenaselösung auf Eis bis zum Gebrauch. </li><li> PharmLyse (Ammoniumchlorid Lysereagenz). </li><li> FACS-Puffer II: Dulbeccos Phosphat-gepufferte Saline CMF + 1% FCS. </li></ul></li><li> Dissection der Brustdrüsen (siehe auch 17). <ul><li> Einschläfern weiblichen Mäusen unter Verwendung von Kohlendioxid (CO 2) Einatmen. </li><li> Auf einem Styropor-Oberfläche, platzieren Sie den Mauszeiger auf den Rücken und Pin fest alle vier Gliedmaßen mit 25-Gauge-Nadeln (Abbildung 1A). Sprühen Sie die Maus großzügig mit 70% Ethanol. </li><li> Mit einer Pinzette, ziehen Sie die Bauchhaut an der Mittellinie und machen einen kleinen Schnitt mit einer scharfen Schere. </li><li> Ausgehend von dem Einschnitt, schneiden die Haut bis zum Hals des Tieres unter Vermeidung Punktieren der Bauch-oder Brusthöhle. </li><li> Schneiden die Haut an den hinteren Beinen von der Mittellinie Einschnitt in Richtung der Schenkel, was zu einer &quot;Y-förmig&quot;. </li><li> Ziehen Sie die Haut vom mouse Körper und festzunageln, Freilegung der Milchdrüsen an der Unterseite der Haut (Abbildung 1B). </li><li> Mit Q-Tips vorsichtig trennen Milchdrüsen von der Haut während des Schneidens Bindegewebe mit kleinen scharfen Schere die Brustdrüse Richtung Wirbelsäule der Maus trennen. </li><li> Die Bauch-Drüsen (# 4, 5) sind am besten für dieses Protokoll. Sie können auch die 2. Brustdrüse, aber bedenken Sie, dass diese Drüsen anatomisch sind in unmittelbarer Nähe zu dem Brustmuskel, die nur schwer zu trennen kann, was in den Zellen des gemischten Gewebe Ursprungs befindet. </li><li> Hinweis: Wenn Sezieren Tumorgewebe, empfiehlt es sich, nekrotische Bereiche zu entfernen. </li></ul></li><li> Dissection des Hautgewebes: Der einfachste Weg, um das Hautgewebe zu erhalten, ohne sich mit Haarentfernung umzugehen ist, Mäuseohren verwenden. Wenn eine größere Menge des Gewebes erforderlich ist, können Sie rasieren Haare von der Maus Oberkörper und sezieren Haut. <ul><li> Einschläfern Mäusen usingen CO 2-Inhalation. </li><li> Mit einer scharfen Schere, schneiden Sie beide Ohren und unmittelbar bei der Verdauung Gefäß mit einem kleinen Volumen (~ 0,5 ml) PBS auf Eis. </li></ul></li></ol> 2. Vorbereitung der Brustdrüse / Haut Single Cell Suspension <ul><li> Zeigen Brustdrüsen / Haut Verdauung Gefäß mit einem kleinen Volumen von PBS auf Eis. Wenn Gewebe blutige Wäsche x2 mit PBS und dann entsorgen überschüssiges PBS. </li><li> Hackfleisch gründlich mit gebogenen Schere. </li><li> 20 ml Kollagenase-Lösung (Anmerkung: diese Menge an Kollagenase-Lösung effizient distanzieren ca. 5 g von Mamma-oder Tumorgewebe und Ohren von bis zu 15 Mäuse). </li><li> Zeigen sofort auf Rührplatte in 37 ° C Wasserbad und inkubieren für 15 min unter Rühren bei mittlerer Geschwindigkeit. </li></ul> Hinweis: optimale Inkubationszeiten kann variieren, wenn verdauen anderen Geweben. <ul><li> Um die Reaktion zu stoppen, fügen Sie 30 ml kaltem DMEM + 10% FCS. </li><li> Der Stamm der Gewebe / Medien Gemisch durch ein 70 um Zellsieb auf einem 50 ml konischen Röhrchen gegeben. </li><li> Zentrifugieren konischen Röhrchen 5 min bei 450 × g bei 4 ° C. </li></ul> 3. Red Blood Cells Lysis Hinweis: Dieser Schritt ist nicht erforderlich, wenn Mäuse wurden mit PBS Herz perfundiert, bevor sie getötet wurden. <ul><li> Überstand unter Verwendung einer Glaspipette, die an Vakuum, ohne das Zellpellet. </li><li> Um RBC, Zellpellet in 10 ml PharmLyse Lösung, Inkubation für 5 min bei Raumtemperatur lysiert. </li><li> Neutralisieren die PharmLyse Lösung durch Zugabe von ca. 40 ml FACS-Puffer I. </li><li> Spin der konischen Rohrs mit Zellen für 5 min bei 450 × g bei 4 ° C. </li><li> Überstand </li></ul> 4. Blockierung der endogenen Fc <ul><li> Optional: Zählen von Zellen auf eine angemessene Lautstärke für folgende ste bestimmenps. </li><li> Die Zellen in 1-3 ml FACS-Puffer I (je nach Zellzahl und Menge des Antikörpers die Sie verwenden möchten). </li><li> Fügen FcBlock (anti-Maus CD16/CD32) bis 1:50 Verdünnung (10 ug / ml) zu erreichen, und auf Eis inkubieren für 10-20 &quot;. Keine Notwendigkeit zu waschen FcBlock. </li></ul> 5. Färbung <ul><li> Nehmen Sie 100 ul Aliquots in Eppendorf-Röhrchen als un-gefärbten Steuerung und einfarbig Kontrollen (nach der Anzahl der fluoreszierenden Antikörpern verwendet wird) verwendet werden. </li><li> Um Label Fibroblasten: add anti-PDGFRα (direkt konjugiert Fluorophor) zu einer Verdünnung von 1:50 (10 ug / ml). </li><li> Um andere Zellpopulationen (zB Immunzellen, Makrophagen, Endothelzellen, etc.) bezeichnen: in geeignete fluoreszenzmarkierte konjugierten Antikörpern gegen Oberflächen-Marker, einer 1:50-Verdünnung. </li></ul> Anmerkung: Obwohl PDGFRα ist eine robuste Marker für Fibroblasten, ist es empfehlenswert, für i Antikörper umfassenmmune Zellen und für Epithelzellen, um etwaige doppelte positive Populationen auszuschließen und somit Verbesserung der Reinheit der isolierten Fibroblasten. <ul><li> Add 2 ul jedes Antikörpers zu jeder der einzelnen Farbregelung Eppendorf-Röhrchen. </li><li> Inkubieren von Zellen mit Antikörpern für 1 Stunde auf Eis im Dunkeln. </li><li> Zum Waschen: Füllen Rohre mit FACS-Puffer I und Spin für 5 min bei 450 xg bei 4 ° C. </li><li> Absaugen des Überstandes, resuspendieren Zellen in FACS Puffer I bis zu einer Konzentration von etwa 2 x 10 6 Zellen / ml, oder welche Konzentration am besten geeignete für die Sortierung mit den verfügbaren FACS Sorter. </li></ul> Optional: Die Zellen können in FACS-Puffer II für die Dauer von Sortier resuspendiert werden. Dies kann die Lebensfähigkeit der Zellen. Jedoch kann die Exposition gegenüber Faktoren in Serum einen Effekt Genexpression, die getestet werden soll und berücksichtigt. <ul><li> Übertragen markierten Zellen in FACS-Röhrchen mit Filter oben: Filter Zellen in den Rohrenverhindern Zellklumpen. </li><li> Um tote Zellen auszuschließen, fügen 7AAD (0,5 ug / ml) oder Propidiumiodid (PI) zu Proben (mit Ausnahme der un-gefärbten Steuerung). </li></ul> Hinweis: Um zu vermeiden, verschwenden Zellen, können Sie 7AAD/PI der un-gefärbten Probe hinzufügen, nachdem die Aufnahme in den FACS, und verwenden Sie wieder als 7AAD nur Kontrolle. <ul><li> Die Proben auf Eis während der Analyse. </li></ul> 6. Isolierung von Zellen durch FACS (Hinweis: dieser Teil des Protokolls erfordert Vorkenntnisse in Betrieb einer FACS-Sorter, zB BD FACS AriaII oder die Hilfe von einem Fachmann). <ul><li> Mit dem FACS Sorter Software (BD FACSDiva), bereiten entsprechende Analysen Plots Vorwärtsstreuung (FSC), seitliche Streulicht (SSC), 7AAD, FITC, PE und andere Fluorophor zur Kennzeichnung Zellen (Abbildung 2) zu analysieren. </li><li> Vor dem Laden jeder Probe in die Zelle Sorter, kurz vortexen zu resuspendieren Zellen. </li><li> Beginnen Sie, indem analyzIng. den ungefärbten Probe, um das Gating der gesamten Zellpopulation gesetzt, um Gate aus Zelltrümmern und um Autofluoreszenz oder Hintergrundfärbung zu bestimmen. </li><li> Analysieren ungefärbten Probe + 7AAD zu bestimmen, und Gate die Population lebender Zellen aus der gesamten Zellpopulation. </li><li> Analysieren Sie jede einzelne Farbe Kontrolle zu bestimmen und zu kalibrieren Parameter wie Entschädigung. </li><li> Analysieren der gefärbten Probe, um die Position von Gattern zum Sortieren definieren. </li><li> Sobald Parameter und Toren für die gewünschten Zellpopulationen gesetzt wurden, beginnen Sortieren der markierten Zellpopulationen in Eppendorf-Röhrchen oder 15 ml konischen Röhrchen mit Kulturmedium oder RNA Lysepuffer (wie RLT-Puffer oder Trizol). </li></ul> Anmerkung: Wenn das Sortieren einer großen Anzahl von Zellen, wird es nicht empfohlen, um direkt zu sortieren in RNA Lysepuffer, wie das große Volumen von Hüllfluid begleitende die Zellen des Lysepuffers verdünnen und die Ausbeute reduzieren. <ul><li> Spin Zellen nach unten unmittelbar nach Sortierung ist abgeschlossen, und die Übertragung in frisches Medium oder RNA Lysepuffer, je nach Zweck des Experiments. </li><li> Wenn Sortieranlagen, um Kultur isolierten Zellen, führen sterile sort. Zur besseren Gewinnung von Zellen, benutzt Phenolrot-freiem DMEM + 5% FCS anstatt FACS-Puffer II und sammeln Zellen in Kulturmedium mit 20% FKS. </li><li> Wenn Kultivierung von Fibroblasten sortiert, auf Kollagen beschichteten Platten alwaysplate und nie direkt auf Kunststoff, da dies zu einer Aktivierung von Fibroblasten führt zu Veränderungen in ihrer Genexpression. </li></ul>

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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. 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Während Versuche in Zellkultur durchgeführt kann informativ und schlagen Funktionsprinzipien, die in vivo nachgewiesen werden kann, ist es bekannt, dass große Veränderungen in der Genexpression von Zellen in Kultur 11,12. Um eine Gewebekultur Schritt vermeiden beim Profilieren Genexpression in Fibroblasten, entwickelten wir ein Protokoll, das Isolierung von normalen, sowie Krebs assoziierten Fibroblasten aus frischen Maus oder menschlichen Gewebe. Ermöglicht Dieses Protokoll wurde erfolgreich mi...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; Name des Reagenz</strong</td&gt;<td<strong&gt; Unternehmen</strong</td&gt;<td<strong&gt; Katalog-Nummer</strong</td&gt;<td<strong&gt; Kommentare (optional)</strong</td&gt;</tr&gt;<tr&gt;<td&gt; DMEM</td&gt;<td&gt; Gibco</td&gt;<td&gt; 41965</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; PBS</td&gt;<td&gt; Biological Industries</td&gt;<td&gt; 02-023-1A</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Collagenase II</td&gt;<td&gt; Worthington</td&gt;<td&gt; LS4176</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; IV Collagenase</td&gt;<td&gt; Worthington</td&gt;<td&gt; LS4188</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Deoxyribonuclease</td&gt;<td&gt; Worthington</td&gt;<td&gt; LS2007</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; PharmLyse</td&gt;<td&gt; BD</td&gt;<td&gt; 555899</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Cell Sieb 70 um</td&gt;<td&gt; SPL</td&gt;<td&gt; 93070</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Purified anti-Maus CD16/CD32</td&gt;<td&gt; BD Pharmingen</td&gt;<td&gt; 553142</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Via Sonde (7AAD)</td&gt;<td&gt; E-Bioscience</td&gt;<td&gt; 00-6993-50</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-Maus-CD140a-PE (PDGFRA)</td&gt;<td&gt; E-Bioscience</td&gt;<td&gt; 12-1401-81</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-Maus-F4/80- FITC</td&gt;<td&gt; Cederlane</td&gt;<td&gt; CL8940F</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; DMEM w / o Phenolrot</td&gt;<td&gt; Gibco</td&gt;<td&gt; 31053</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Kollagen Typ I</td&gt;<td&gt; BD Biosciences</td&gt;<td&gt; 354236</td&gt;<td</td&gt;</tr&gt;</tbody&gt;</table

isolation of normal and cancer-associated fibroblasts from fresh tissues by fluorescence activated cell sorting (facs)

Krebs-assoziierte Fibroblasten (CAF) sind die bekanntesten Zelltyp innerhalb des Tumorstroma vieler Krebsarten, insbesondere Mammakarzinomen, und ihre höchste Präsenz wird oft mit einer schlechten Prognose assoziiert 1,2. CAF sind aktiviert Subpopulation von Stromafibroblasten, Ausdruck von denen viele die Myofibroblasten Marker α-SMA 3. CAF stammen vom lokalen Gewebe Fibroblasten sowie aus Knochenmark stammende Zellen rekrutiert in die sich entwickelnde Tumoren und Annahme eines CAF Phänotyp unter dem Einfluß des Tumormikroumgebung 4. CAF wurde gezeigt, dass Tumorinitiation, Wachstum und die Progression durch signalisiert, dass fördert die Proliferation von Tumorzellen, die Angiogenese und Invasion 5-8 erleichtern. Wir haben gezeigt, dass CAF Tumorwachstum verstärken durch Vermittlung tumorpromovierenden Entzündungen, ab den frühesten präneoplastische Stufen 9. Trotz der zunehmenden Nachweis der Schlüsselrolle CAF spielen bei der Förderung des Tumorwachstums,Studium CAF wurde ein fortlaufender Herausforderung aufgrund der mangelnden CAF-spezifische Marker und die überwiegende Heterogenität dieser Zellen, mit vielen Subtypen koexistierenden im Tumormikromilieu 10. Außerdem prüft Fibroblasten in vitro durch die Tatsache, dass ihre Genexpressionsprofils oft in Gewebekultur 11,12 verändert behindert. Um dieses Problem anzugehen und unvoreingenommene Genexpressionsanalysen von Fibroblasten aus frischen Maus und menschlichen Geweben zu ermöglichen, entwickelten wir ein Verfahren bei früheren Protokollen für Fluorescence-Activated Cell Sorting (FACS) 13,14 basieren. Unser Ansatz beruht auf der Nutzung PDGFRα als Oberflächenmarker zu Fibroblasten aus frischen Maus und menschlichem Gewebe zu isolieren. PDGFRα ist reichlich von beiden normalen Fibroblasten und CAF 9,15 ausgedrückt. Diese Methode erlaubt die Isolierung von reinem Populationen von normalen Fibroblasten und CAF, einschließlich, jedoch nicht darauf beschränkt α-SMA + aktiviert Myofibroblasten. Isolierte Fibroblasten können dannverwendet für die Charakterisierung und Vergleich der Entwicklung der Genexpression, die in der Tumorgenese CAF auftritt. Tatsächlich berichteten wir und andere Expression Profiling von Fibroblasten durch Zellsortierung 16 isoliert. Dieses Protokoll wurde erfolgreich durchgeführt zu isolieren und zu profilieren hochangereichertes Populationen von Fibroblasten aus der Haut, Brust-, Pankreas-und Lungengewebe. Außerdem unserer Methode können auch Kultivieren von sortierten Zellen, um funktionelle Experimente durchzuführen und um die Kontamination durch Tumorzellen, die oft eine große Hürde beim Versuch Kultur CAF vermeiden.

Verwendung PDGFRα als Marker für Fibroblasten führt Isolierung von hochangereicherten Populationen von Gewebe Fibroblasten. Der Reinheitsgrad nach der Sortierung betrug 99%, wie durch post-sort-Analyse (2A) quantifiziert. Schätzen der Prozentsatz der nicht-kontaminierenden Fibroblastenzellen durch die relative Expression von zellspezifischen Kontrollgene (2B) zeigt typischerweise 0,1-0,6% Verunreinigung. Diese Reinheit ermöglicht hohe Qualität Transkriptom-Profiling von isolierten...

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

Krebs Fibroblasten (CAF) zu erleichtern Tumorinitiation, Wachstum und die Progression durch signalisiert, dass es fördert die Proliferation, Angiogenese und Entzündung. Hier beschreiben wir ein Verfahren, um reine Populationen normalen Fibroblasten und CAF aus frischen Maus und menschlichen Geweben isolieren durch Zellsortierung unter Verwendung PDGFRα als Oberflächen-Marker.

Isolation von Normal und Krebs-assoziierte Fibroblasten von Fresh Gewebe durch 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 ...

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Biology

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

35.5K Aufrufe.  Tel Aviv University. Krebs Fibroblasten (CAF) zu erleichtern Tumorinitiation, Wachstum und die Progression durch signalisiert, dass es fördert die Proliferation, Angiogenese und Entzündung. Hier beschreiben wir ein Verfahren, um reine Populationen normalen Fibroblasten und CAF aus frischen Maus und menschlichen Geweben isolieren durch Zellsortierung unter Verwendung PDGFRα als Oberflächen-Marker.

Serie: 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.

Isolierung und Reinigung von Drosophila Peripheren Neuronen durch Magnetic Bead Sorting

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

Forschung

Biologie

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.

Fluorescence Activated Cell Sorting von Pflanzenprotoplasten

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.

Bromodeoxyuridine (BrdU) Labeling und anschließende Fluorescence Activated Cell Sorting für Kultur-unabhängige Identifizierung von gelöstem organischem Kohlenstoff abbauenden Bakterioplankton

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.

Isolierung von Ratten-Portal Fibroblasten durch<em&gt; In-situ</em&gt; Leberperfusion

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.

Zellspezifische Analyse<em&gt; Arabidopsis</em&gt; Blätter mit 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 ...

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.

Analyse von Zellsuspensionen isoliert von festen Geweben von 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 ...

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.

Magnetische aktiviert Zelle Sortieren Strategien zu isolieren und reinigen Synovial Flüssigkeit abgeleitet mesenchymalen Stammzellen aus einem Kaninchen-Modell

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.

Single Cell Micro-Aspiration als Alternative zur fluoreszenzaktivierten Zellsortierung für die Trennung von Riesenviren

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.

Fluoreszenz-aktivierte Zellsortierung zur Isolierung von Skleractin-Zellpopulationen

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.

Isolierung und Charakterisierung von Exosomen aus Skelettmuskelfibroblasten

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