Die Finanzierung dieses Projekts wurde von der National Science Foundation gewährt OCE1029607 (XM) und MCB0702125 (MAM) und Gordon und Betty Moore Foundation (MAM) zur Verfügung gestellt.

1. Wasserprobe Verarbeitung <ol><li> Filter 10L Umwelt Wasser durch 1 &amp;mgr; m-Porengröße Membranfilter zur Entfernung großer Partikel und bacteriovores. Sammeln Sie die Wasser-Filtrat in einem Kanister. </li><li> Transfer 36 ml Filtrat in je 3 sterile Eppendorf-Röhrchen (50 ml) mit 4 ml frisch zubereitete Paraformaldehyd-Lösung (PFA; 10%). Inkubieren für 2 Stunden bei Raumtemperatur, um Zellen zu erhalten. Sammeln Zellen auf 0,22-um-Porengröße weiß Membranfilter durch Vakuumfiltration. Waschen Sie den Filter, indem 10 ml Kochsalzlösung (PBS) Phosphat-gepufferte durch den Filter durch Vakuumfiltration. Beschriften Sie die Filter als negative Kontrollen und lagern Sie sie bei -20 ° C. </li></ol> Steps 1,3-1,4 sind für die Errichtung DOC-limitierten Bedingungen optional. <ol start="3"><li> Eine Mischung von anorganischem Stickstoff und Phosphor (5 pM NH 4 Cl, 5 uM NaNO 3 und 1 &amp;mgr; M NaH 2 PO 4 Endkonzentration) in den Kanister. </li><li> Inkubieren im Dunkeln bei in situ Temperatur für 48 Stunden mit gelegentlichen Erschütterungen. </li></ol> 2. Mikrokosmos Einrichtung und Inkubation <ol><li> Füllen Sie jede der sechs 1-L Glasflaschen mit 800 ml Wasserprobe aus dem Ballon der Schritt von 1,4 bis Mikrokosmen zu etablieren. Add BrdU (10 uM, Endkonzentration) zu jeder Mikrokosmos. Gut mischen. </li><li> 1 ml Modell DOC Verbindung Lösung in drei der Mikrokosmen, werden diese als DOC Änderungen dienen. 1 ml sterilem PBS in den verbleibenden drei Mikrokosmen, werden diese als nicht-Addition Kontrollen dienen. </li><li> Inkubieren Sie alle Mikrokosmen in einem Inkubator Schüttler inkubieren im Dunkeln bei in situ Temperatur unter Schütteln bei 100 Umdrehungen pro Minute. </li><li> Sammeln Sie 36 ml Wasser Probe von jeder Mikrokosmos und Transfer in 50 ml sterile Eppendorf-Röhrchen zu den Zeitpunkten 0, 8, 16, und 24 Stunden. Unmittelbar 4 ml frisches PFA (10%) auf Sammelröhrchen und Inkubation für 2 Stunden bei Raumtemperatur, um die Zellen zu erhalten. </li><li> Filter bewahrt Zellen durch 0,22-um-Porengröße Polykarbonatmembranfilter. Waschen Sie den Filter mit 10 ml PBS. Gehen Sie sofort zum nächsten Schritt oder lagern Sie die Filter bei -20 ° C. </li></ol> 3. In situ Immundetektion für BrdU-Einbau <ol><li> Thaw die Filter (DOC geändert Proben, ohne zusätzlich Kontrollen und negative Kontrollen) bei Raumtemperatur. </li><li> Es wird 1 ml Lysozym-Lösung [10 mg / ml Lysozym Eiweiß in 100 mM Tris, 50 nM EDTA (pH = 8)] 4 bis Bakterienzellen auf dem Filter zu decken. Inkubieren bei Raumtemperatur für 30 Minuten. Waschen Sie den Filter, indem 10 ml PBS durch sie unter Absaugung. </li><li> 1 ml Proteinase K-Lösung [2 mg / ml Proteinase K in 100 mM Tris, 50 nM EDTA (pH = 8)] 4 bis Bakterienzellen auf dem Filter zu decken. Inkubieren bei Raumtemperatur für 30 Minuten. Waschen Sie den Filter, indem 10 ml PBS durch sie unter Absaugung. </li><li> 1 ml Exonuklease-Lösung [Exonuklease III (50 U / ml) in 5 mM MgCl 2 und 50 mM Tris-HCl] 5 bis Bakterienzellen auf dem Filter zu decken. Bei 37 ° C für 30 Minuten. Waschen Sie den Filter, indem 10 ml PBS durch sie unter Absaugung. </li><li> Montieren Sie frame-Siegel Inkubationskammern (Bio-Rad) nach den Anweisungen des Herstellers. </li><li> Schneiden Sie die Filter in Achtel mit einer sterilen Klinge auf ein Alkohol gereinigt trockene Oberfläche. </li><li> Mit einer Pinzette, legen ein achter Abschnitt eines Filters zu einem montierten Rahmen-Dichtung Inkubationskammer (acht frame-Siegel Kammern sind für jeden Filter Probe erforderlich). Die Seite der Filter-Sektion, die Zellen enthält, sollte nach oben zeigen. Die Feuchtigkeit an der Rückseite des Filters wird der Filter-Stick, um die Folie zu ermöglichen. Wenn der Filter trocken wird, wenden Sie einen kleinen Tropfen (2 ul) diH 2 O in der Mitte der Kammer, bevor der Filter-Sektion auf der Folie. </li></ol> Steps 3,8-3,20 Verwendung der Reagenzien aus den ROCHE In Situ Cell Proliferation Kit, FLUOS folgenden Verfahren aus der Anleitung des Herstellers modifiziert. Mit Ausnahme von PBS, sind alle Reagenzien im Kit enthalten. <ol start="8"><li> Genügend Inkubationspuffer (0,5% Rinderserumalbumin, 0,1% Tween20 in PBS,) gleichmäßig auf die gesamte Filter-Sektion in der Inkubationskammer ohne Überlauf, wenn die Dichtung aufgebracht wird. </li><li> Legen Sie eine Polyester-Frame Abdeckung über dem Brutraum Rahmen. Drücken Sie fest zu verschließen Inkubationskammer. Vermeiden Sie Luftblasen über dem Filter. Inkubieren Sie die verschlossenen Kammern für 10 min bei Raumtemperatur im Dunkeln. </li><li> Entfernen Sie die Polyester-Siegel und öffnen Sie die Inkubationskammern. (Hinweis: Öffnen der Kammern manchmal wird hochziehen Rahmen Dichtung aus der Folie in diesem Fall, bereiten Sie einen neuen Raum für die folgenden Schritte..) </li><li> Pipettieren Sie die Inkubationspuffer von einer Ecke der Kammer. Vermeiden Sie den Kontakt mit dem Filter. </li><li> Waschen Sie den Filter durch leichtes Pipettieren 100 ul PBS in undaus der Kammer 3 mal. </li><li> Bereiten anti-BrdU-FLUOS funktionierende Lösung die folgenden Schritte durch den Hersteller unmittelbar vor Gebrauch empfohlen. </li><li> Pipet 120 ul Anti-BrdU-FLUOS funktionierende Lösung auf den Filter, wobei darauf zu gleichmäßig auf die gesamte Oberfläche. </li><li> Verschließen Sie die Kammer mit einer neuen Polyester-Bezug. Vermeiden Sie Luftblasen auf der Oberseite des Filters. Inkubieren Sie die Kammer im Dunkeln bei 37 ° C für 3 Stunden. Dieser Schritt wird BrdU eingebaut DNA in situ mit Fluoresceinisothiocyanat (FITC)-Label. </li><li> Entfernen Sie die Polyester-Abdeckung und öffnen Sie die Inkubationskammer. Pipettieren aus der Anti-BrdU-FLUOS funktionierende Lösung. Waschen Sie den Filter 3 mal mit PBS. </li><li> Übertragen Sie die Filter aus dem Brutraum in eine sterile Oberfläche. Schneiden Sie die Filter-Sektion in kleine Stücke mit einer sterilen Klinge. </li><li> Übertragen Sie die Filter Stücke in 2 ml Reaktionsgefäße mit je 1 ml PBS. Fest verschließen den Schlauch und Dichtung mit Parafilm. Bei 37 ° C und 200 rpm für 10 min. </li><li> Befestigen Sie die Schläuche auf einem Vortexer und Vortex bei maximaler Geschwindigkeit für 5 Minuten. Pipettieren der Überstand in ein steriles 15 ml Eppendorf-Röhrchen. Wiederholen Sie die Inkubation und Wirbel Schritte für 5 weitere Male. Kombinieren Sie den Überstand in den gleichen 15 ml Eppendorf-Röhrchen für jede Probe. In der Regel können 80% Zellen in der Suspension zurückgewonnen werden. </li><li> Bewahren Sie den Überstand mit resuspendierten Zellen bei 4 ° C. Sortieren innerhalb von 2 Tagen. </li></ol> 4. FACS-Analyse Ein Verfahren für eine BD FACSAria Durchflusszytometer und entsprechender Software wird hier beschrieben. <ol><li> Optimieren Sie die Einstellung der Durchflusszytometer folgenden Schritte vom Hersteller empfohlen. Dies bedeutet: Einstellung der Verstärkung, um die gewünschte breakoff Parameter und den Sweet Spot gesetzt; Optimierung der Laser-Verzögerung und Umgebung Skalierungsfaktoren für das Experiment Mantel Druck und Optimierung der Einstellungen für FSC und SSC Spannungen, FSC Schwelle, FSC-Fluoreszenz-Skalierung, Fluoreszenz-PMT-Spannungen , etc. </li><li> Run negativen Kontrollproben am Durchflusszytometer (FCM) auf Fluoreszenz-Intensität des FITC und side scatter (SSC) basiert. Erhöhen Sie die FITC Schwelle, bis keine Zellen in den negativen Kontrollen können visualisiert werden durch die FITC-SSC Erwerb anzuzeigen. </li><li> Run no-Neben Kontrollproben und untersuchen das Verteilungsmuster von 10.000 Zellen auf FITC und SSC Übernahmen. Definieren Sie ein Tor, um alle Zellen umschließen und bezeichnen sie als &quot;low intensity Zellen&quot; (LIS). </li><li> Run DOC-Fassung Proben und untersuchen das Verteilungsmuster von 10.000 Zellen auf FITC und SSC Basis. Einige Zellen werden in der vorgegebenen LI Tor erscheinen. Definieren Sie ein weiteres Tor zu den Zellen, die höhere Intensität der Fluoreszenz (HIS) als LIs haben umschließen. Erwerben Sie Statistiken, um die relative Fülle von gated Zellen anzuzeigen. </li><li> Sortieren HALLO Zellen in Sammelröhrchen mit 500 ul PBS bei &quot;reinigen 1 Tropfen&quot;-Modus. Terminate Sortierung, wenn die Zahl seiner erreicht 500.000 zählt. </li></ol> 5. Filter PCR-Amplifikation von 16S rRNA-Gene Filter PCR-Verfahren werden aus Kirchman et al geändert. 6 <ol><li> Filter sortierten Zellen auf eine weiße, 0,22 um-Porengröße, 25 mm Durchmesser, Polycarbonat-Membranfilter. Schneiden Sie den Rand des Filters, dass keine bakterielle Zellen mit einer sterilen Klinge enthält. Schneiden Sie die Filter in 8 gleich große Stücke schneiden. </li><li> Legen Sie eine einzelne Filter Stück in ein PCR-Gefäß, mit dem Zellen nach innen der Röhre. Add 45 ul PCR-Wasser in die PCR-Reaktionsgefäß. Tauchen Sie den Filter komplett im Wasser. </li><li> Add 2 ROCHE Abbildungen PuReTaq Read-To-Go PCR Beads in die PCR-Gefäß, kurz vortexen. Add 2 ul jeder der Vorwärts-und Rückwärtsfahrt 16S rRNA-Gen-Primer (0,4 uM Endkonzentration für jedes Primer), wie 27F und 1492R 7, in jedem der PCR Reaktionsgefäße. </li><li> (Optional) Fügen 1μl Rinderserumalbumin-Lösung (BSA, Endkonzentration 30μg/100 ul) der PCR Reaktionsmischung zu adsorbieren Verstärkung Hemmer helfen. Wenn man nicht an BSA hinzufügen wählt, fügen 1μl Wasser statt. </li><li> Führen Sie die PCR-Amplifikation auf einem Thermocycler. A touch down PCR-Programm empfohlen wird, hat die Glühtemperatur sequenziell abnehmenden von 62 bis 52 ° C um 1 ° C pro Zyklus für 11 Zyklen von 15 Zyklen mit Annealingtemperatur von 52 ° C, gefolgt Alle Zyklen sind Denaturierung (bei 95 ° C), Annealing (at 62 bis 52 ° C) und Verlängerung (bei 72 ° C) Schritte der 50er Jahre Dauer. Eine erste 3-min Denaturierung und letzte 10-min-Erweiterung wird auch für die PCR-Programm enthalten. </li><li> Bestätigen Sie die PCR-Amplifikation mittels Elektrophorese auf einem mit Ethidiumbromid gefärbten 1% Agarosegel. Excise der PCR-Produkte aus dem Gel und sauber mit dem QIAGEN QIAquick Gel Extraction Kit. </li><li> Führen Sie zwei zusätzliche PCR-Amplifikationen für jede Probe, verwenden Sie jedes Mal eine neue Filter-Sektion. Nach PCR-Gel purificatiauf, bündeln die Amplikons der gleichen Probe zusammen. Gereinigtes 16S rDNA Amplikons sind nun bereit für eine Reihe von molekularen Analysen, taxonomische Identifizierung, wie Klon-Bibliothek Bau-und Sequenzierung zu ermöglichen. </li></ol> 6. Repräsentative Ergebnisse: Repräsentative Ergebnisse werden anhand einer Studie von Putrescin-abbauenden Bakterien als ein Beispiel. Wasserproben wurden von einem Küstengebiet von Georgien gesammelt und verarbeitet nach dem oben beschriebenen Verfahren. FACS-Analyse ergab, dass Putrescin Neben der Entwicklung einer Gruppe von Bakterien mit hohem FITC Fluoreszenz-Intensität induziert, was eine hohe BrdU-Einbau-Rate (Abbildung 1). Diese Zellen wurden als High-BrdU-Einbau-Zellen (HIS) bezeichnet und wurde erwartet, enthalten meist Putrescin-abbauenden Bakterien. Seine waren in der nicht-Addition Kontrollen, die nur enthalten Zellen mit niedrigerem BrdU-Einbau (LIS) fehlt. LIs wurde erwartet, dass vor allem enthalten Bakterioplankton, dass nicht hinzugefügt Putrescin Einsatz waren. His wurden in getrennten Röhren sortiert und dann gesammelt auf Membranfiltern. 16S rRNA-Gen Amplikons wurden für hohe HALLO Zellen mittels Filter PCR erhalten. <img src="/files/ftp_upload/2855/2855fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Durchflusszytometrische Analyse von nicht-zusätzlich-Steuerung und Modell-Compound-Fassung (Putrescin hier als Beispiel) Proben nach 24 h Inkubation gesammelt. Zellverteilung Analyse wurde auf (1) Fluoreszenzintensität FITC-Markierung (x-Achse), was sich positiv auf Ebene des BrdU-Einbaus ist im Zusammenhang basiert, und (2) side scatter (SSC, y-Achse), die positiv zu Zelle im Zusammenhang Größe. Tor Notation ist auf Ebene des BrdU-Einbaus, (; LI, low-BrdU-Inkorporation HALLO, High-BrdU-Inkorporation) basiert. Der relative Anteil der HALLO-und LI-Zellen sind in entsprechenden Toren gezeigt.

<ol>
	<li>Pernthaler, A., Pernthaler, J., Schattenhofer, M., Amann, 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+DNA-synthesizing+bacterial+cells+in+coastal+North+Sea+plankton.">Identification of DNA-synthesizing bacterial cells in coastal North Sea plankton.</a> Appl. Environ. Microbiol. 68, 5728-5728 (2002).</li><li>Urbach, E., Vergin, K. L., Giovannoni, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunochemical+detection+and+isolation+of+DNA+from+metabolically+active+bacteria.">Immunochemical detection and isolation of DNA from metabolically active bacteria.</a> Appl. Environ. Microbiol. 65, 1207-12 (1999).</li><li>Mou, X. Z., Hodson, R. E., Moran, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterioplankton+assemblages+transforming+dissolved+organic+compounds+in+coastal+seawater.">Bacterioplankton assemblages transforming dissolved organic compounds in coastal seawater.</a> Environ. Microbiol. 9, 2025-2025 (2007).</li><li>Hodson, R. E., Dustman, W. A., Garg, R. P., Moran, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+PCR+for+visualization+of+microscale+distribution+of+specific+genes+and+gene+products+in+prokaryotic+communities.">In situ PCR for visualization of microscale distribution of specific genes and gene products in prokaryotic communities.</a> Appl. Environ. Microbiol. 61, 4074-4074 (1995).</li><li>Dinjens, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bromodeoxyuridine+(BrdU)+immunocytochemistry+by+exonuclease+III+(Exo+III)+digestion.">Bromodeoxyuridine (BrdU) immunocytochemistry by exonuclease III (Exo III) digestion.</a> Histochemistry. 98, 199-199 (1992).</li><li>Kirchman, D. L., Yu, L. Y., Fuchs, B. M., Amann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+bacterial+communities+in+aquatic+systems+as+revealed+by+filter+PCR.">Structure of bacterial communities in aquatic systems as revealed by filter PCR.</a> Aquat. Microb. Ecol. 26, 13-13 (2001).</li><li>Delong, E. F., Wickham, G. S., Pace, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+stains:+ribosomal+RNA-based+probes+for+the+identification+of+single+cells.">Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells.</a> Science. 243, 1360-1360 (1989).</li><li>Artursson, V., Jansson, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+bromodeoxyuridine+immunocapture+to+identify+active+bacteria+associated+with+arbuscular+mycorrhizal+hyphae.">Use of bromodeoxyuridine immunocapture to identify active bacteria associated with arbuscular mycorrhizal hyphae.</a> Appl. Environ. Microbiol. 69, 6208-6208 (2003).</li><li>Mou, X. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+carbon+processing+by+generalist+species+in+the+coastal+ocean.">Bacterial carbon processing by generalist species in the coastal ocean.</a> Nature. 451, 708-708 (2008).</li><li>Mou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-cytometric+cell+sorting+and+subsequent+molecular+analyses+for+culture-independent+identification+of+bacterioplankton+involved+in+dimethylsulfoniopropionate+transformations.">Flow-cytometric cell sorting and subsequent molecular analyses for culture-independent identification of bacterioplankton involved in dimethylsulfoniopropionate transformations.</a> Appl. Environ. Microbiol. 71, 1405-1405 (2005).</li><li>Dean, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+human+genome+amplification+using+multiple+displacement+amplification.">Comprehensive human genome amplification using multiple displacement amplification.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 5261-5261 (2002).</li></ol>

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Unser Ansatz Paare BrdU-Einbau, FACS und 16S rDNA-Analyse zu ermöglichen Arten-Identifikation auf der Bakterioplankton, dass die einzelnen Komponenten DOC verstoffwechseln in der aquatischen Umwelt. Die BrdU-Einbau-Assay Etiketten Bakterienzellen auf Stoffwechselaktivitäten, die Analyse erlaubt nur auf aktive Bakterien und somit nicht enthalten ruhenden Zellen. In unserem Ansatz wird BrdU-Einbau in situ immunodetected und Bakterien, die verschiedenen Ebenen des BrdU-Einbaus haben, sind anschließend visualisi...

<table border="1"><tbody><tr><td> Name </td><td> Typ </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> BrdU </td><td> Reagens </td><td> Sigma </td><td> B5002-5G </td><td></td></tr><tr><td> Lysozym </td><td> Reagens </td><td> Sigma </td><td> L6876-5G </td><td></td></tr><tr><td> Proteinase K </td><td> Reagens </td><td> Sigma </td><td> P2308-25mg </td><td></td></tr><tr><td> In Situ Cell Proliferation Kit, FLUOS </td><td> Satz </td><td> Roche </td><td> 11810740001 </td><td> Verbrauchen Sie mehr von Anti-BrdU-FLUOS und Inkubationspuffer pro Reaktion als vom Hersteller empfohlen. </td></tr><tr><td> Frame-Seal Inkubationskammern </td><td> Material </td><td> Bio-Rad </td><td> SLF-1201 </td><td></td></tr><tr><td> Polycarbonat-Membranfilter (142-mm-Durchmesser, 1,0 &amp;mgr;-Porengröße) </td><td> Material </td><td> Millipore </td><td> FALP14250 </td><td></td></tr><tr><td> Polykarbonatmembranfilter (25-mm-Durchmesser, 0,2 mu m-pore-size) </td><td> Material </td><td> Millipore </td><td> FGLP02500 </td><td></td></tr><tr><td> Abbildungen PuReTaq Ready-To-Go PCR Beads </td><td> Satz </td><td> GE Healthcare </td><td> 27-9559-01 </td><td></td></tr><tr><td> QIAquick Gel Extraction Kit </td><td> Satz </td><td> QIAGEN </td><td> 28704 </td><td></td></tr><tr><td> FailSafe PCR-System </td><td> Satz </td><td> EPICENTRE </td><td> FS99060 </td><td></td></tr></tbody></table>

bromodeoxyuridine (brdu) labeling and subsequent fluorescence activated cell sorting for culture-independent identification of dissolved organic carbon-degrading bacterioplankton

Mikroben sind wichtige Mittel der Vermittlung der Abbau von zahlreichen gelösten organischen Kohlenstoffs (DOC) Substrate in der aquatischen Umwelt. Allerdings stellt Identifizierung von Bakterien-Taxa, dass bestimmte Pools von DOC in der Natur verwandeln eine technische Herausforderung. Hier beschreiben wir einen Ansatz, Paare Bromdesoxyuridin (BrdU) Inkorporation, Fluoreszenz-activated cell sorting (FACS) und 16S rRNA-Gen-basierte molekulare Analyse, dass die Kultur-unabhängige Identifizierung Bakterioplankton, die einen Abbau einer bestimmten DOC Verbindung in der aquatischen Umwelt ermöglicht. Triplicate Bakterioplankton Mikrokosmen sind bis zu beiden BrdU und ein Modell DOC Verbindung (DOC Änderungen), oder nur BrdU (no-Neben-Steuerung) zu empfangen. BrdU ersetzt die Positionen von Thymidin in neu synthetisierte Bakterien-DNA und BrdU-markierte DNA kann leicht immunodetected 1,2 sein. Durch eine 24-Stunden-Inkubation Bakterioplankton, dass in der Lage, die hinzugefügt DOC Verbindung erwartet werden, um selektiv aktiviert werden, und haben daher ein höheres BrdU-Einbau (HALLO-Zellen) als non-responsive Zellen in der DOC Änderungen und Zellen in nicht-sind Zusätzlich steuert (low BrdU-Einbau-Zellen, LI-Zellen). Nach Fluoreszenz Immundetektion sind HALLO Zellen unterschieden und körperlich von der LI-Zellen durch Fluoreszenz-aktivierte Zellsortierung (FACS) 3 getrennt. Sortiert DOC-responsive Zellen (HALLO-Zellen) sind für die DNA extrahiert und taxonomisch durch nachträgliche 16S rRNA-Gen-Analysen, einschließlich PCR, Klon-Bibliothek Bau-und Sequenzierung identifiziert.

Watch this Scientific Journal Video about Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degradin

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

Environmental Bakterioplankton sind mit einem Modell des gelösten organischen Kohlenstoffs (DOC)-Verbindung und einer DNA Markierungsreagens, Bromdesoxyuridin (BrdU) inkubiert. Anschließend werden DOC-abbauenden Zellen aus dem Bulk-Community auf ihre erhöhte BrdU-Einbaus mittels Fluoreszenz-activated cell sorting (FACS) basiert getrennt. Diese Zellen werden dann durch nachfolgende molekulare Analysen identifiziert.

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 ...

bromodeoxyuridine-brdu-labeling-subsequent-fluorescence-activated

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

20.8K Aufrufe.  Kent State University. Environmental Bakterioplankton sind mit einem Modell des gelösten organischen Kohlenstoffs (DOC)-Verbindung und einer DNA Markierungsreagens, Bromdesoxyuridin (BrdU) inkubiert. Anschließend werden DOC-abbauenden Zellen aus dem Bulk-Community auf ihre erhöhte BrdU-Einbaus mittels Fluoreszenz-activated cell sorting (FACS) basiert getrennt. Diese Zellen werden dann durch nachfolgende molekulare Analysen identifiziert.

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

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

Generation of Human CD40-aktivierten B-Zellen

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

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 ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Lipidvesikel-vermittelte Affinitätschromatographie unter Verwendung von Magnetic Activated Cell Sorting (LIMACS): Eine neue Methode zur Protein-Lipid-Wechselwirkungen Analyze

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

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 ...

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.

Optimiert Färbung und Proliferation Modeling-Methoden für die Zellteilung Überwachung mit 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 ...

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.

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 ...

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the plasma membrane and the Golgi. As a result the endosomal system plays a central role in maintaining cell homeostasis, and mutations in genes belonging to this network of organelles interconnected by vesicular transport, cause severe pathologies including cancer and neurobiological disorders. It is therefore of prime relevance to understand the mechanisms underlying the biogenesis and organization of the endosomal system. The yeast Saccharomyces cerevisiae has been pivotal in this task. To specifically label and analyze at the ultrastructural level the endosomal system of this model organism, we present here a detailed protocol for the positively charged nanogold uptake by spheroplasts followed by the visualization of these particles through a silver enhancement reaction. This method is also a valuable tool for the morphological examination of mutants with defects in endosomal trafficking. Moreover, it is not only applicable for ultrastructural examinations but it can also be combined with immunogold labelings for protein localization investigations.

Yeast, Saccharomyces cerevisiae, has been a key model organism to identify and study genes regulating the biogenesis and functions of the endosomal system. Here we present a detailed protocol for the specific labeling of the endosomal compartments for ultrastructural studies.

Nanogold Markierung der Hefe Endosomale System für Ultrastrukturanalysen

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Sequenzspezifische Markierung von Nukleinsäuren und Proteinen mit Methyltransferasen und Cofaktor-Analoga

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

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 ...