Wir danken Mitarbeiter am Anatomischen Pathologie Laboratories des Royal Prince Alfred und Concord Rückführung Krankenhäuser zum Sammeln frischer Proben von CRC und normalen Darmschleimhaut. Die Arbeit wurde durch einen Cancer Institute New South Wales Translational-Zuschüssen finanziert.

<img src="/files/ftp_upload/3322/3322fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Arbeitsablauf für die Herstellung einer Suspension von lebenden Zellen aus einem chirurgischen Stichprobe von CRC. 1. Klinische Probe Disaggregation Alle Proben wurden von der Royal Prince Alfred Hospital (Camperdown, NSW, Australien) und Concord Rückführung Hospital (Concord West, NSW, Australien) mit Einwilligung im Rahmen des Protokolls Nr. X08-164 gesammelt. <ol><li> Sammeln Sie frische Kolorektalkarzinom (CRC) oder Adenom Proben und normalen Darmschleimhaut mindestens 10 cm aus dem Tumor. Shop-Proben in balanced salt Hank-Lösung pH 7,3 (HBSS) bei 4 ° C für bis zu 12 h nach der Resektion. </li><li> Follow Sicherheitsvorschriften für den menschlichen Krankheitserreger, verarbeiten alle klinischen Proben in einer biologischen Sicherheitswerkbank der Klasse II. Präparieren Sie die Proben in 2 mm Würfel in einer Petrischale mit zwei Skalpellklingen. </li><li> Inkubieren Tumor-und Normalgewebe in separaten Eppendorf-Röhrchen mit gelegentlichen vorsichtig mischen und 60 min bei 37 ° C mit dem gleichen Volumen RPMI 1640-Medium mit 2% (v / v) Kollagenase Typ 4 (Worthington, Lakewood, NJ, USA) und 0,1 % (w / v) Desoxyribonuclease I aus Rinder-pancrease (DNAse I; Sigma-Aldrich). </li><li> Force-Gewebe durch ein feines Drahtgeflecht Sieb mit einem Kolben aus einer 10 ml-Spritze halb verdaute; waschen Sie die Zellen durch mit HBSS. </li><li> Pass resultierende Zellsuspension bis 200 um und 50 um Filcon Filter (BD Biosciences), um Zellaggregate zu entfernen. Die meisten der DNA, Schleim und Zell-Aggregate sind in dieser Reihe von Filtrationen entfernt. </li><li> Centrifuge Zellsuspensionen bei 400 xg bei 20 ° für 5 min. </li><li> Resuspendieren Zellpellets in hitzeinaktiviertem FCS mit 10% Dimethylsulfoxid (DMSO), langsam einfrieren in Kryoröhrchen und bei -80 °. Das Einfrieren Prozess neigt dazu, Schleim in der Probe zu reduzieren und Lyse roter Blutkörperchen. </li></ol> 2. Probenvorbereitung für die Zelle zu erfassen <ol><li> Auftauen Proben schnell in einem 37 ° Wasserbad und die Zellen in 10 ml HBSS zum Auswaschen der DMSO. </li><li> Centrifuge Zellsuspensionen bei 410 xg bei 20 ° für 5 min. </li><li> Den Überstand und Zellpellet in 500 ul HBSS. </li><li> Behandeln Sie die Probe mit 0,1% (w / v) DNAse I für 20 min bei Raumtemperatur. </li><li> Mix 10 ul jeder Zellsuspension mit dem gleichen Volumen Trypanblau und Last 10 ul der Mischung in einer Zählkammer. Mit einem Lichtmikroskop bei 100-facher Vergrößerung, count lebensfähigen Zellen, die deutlich durch Trypanblau-Ausschluss erscheinen, während tote Zellen nehmen den Farbstoff. Ein mindestens 4 x 10 6 lebensfähige Zellen ist für die Zelle zu erfassen auf dem Mikroarray erforderlich. </li><li> Nach DNAse Behandlung, resuspendieren die Zellsuspension in 10 mL HBSS und Zentrifuge bei 410 xg bei 20 ° für 5 min. </li><li> Dekantieren der Überstände und resuspendieren Zellpellets in RPMI 1640 auf ein Volumen von 200 ul. </li></ol> 3. Antikörper Microarray-Zelle zu erfassen <ol><li> Feuchten Sie die DotScan Antikörper Microarray durch Eintauchen der Nitrozellulose Abschnitt in Phosphat-gepufferter Kochsalzlösung (PBS) für etwa 20 s. Wischen Sie die Glaskanten des Mikroarrays mit gefalteten Kimwipes, Vermeidung Berühren Sie die Nitrozellulose Abschnitt. </li><li> Wasser hinzu, bis die Microarray Inkubationswanne um eine feuchte Kammer stellen. Setzen Sie den Microarray in die Kammer und Pipette wurde die Zellsuspension in RPMI 1640 auf die feuchte Nitrocellulose Abschnitt. Pipette Tropfen an jeder Ecke der Nitrocellulose, um sicherzustellen, eine noch von Zellen verteilt. </li><li> Inkubieren Sie die Microarrays bei 37 ° C für 1 h. Die Inkubationszeit kann Zellen, sich niederzulassen und in Kontakt kommen mit Antikörpern auf dem Microarray. Zellen, die Oberflächen-Antigene entsprechend der Antikörper landen sie auf aufgenommen wird. </li><li> Nach der Inkubation, tauchen die Microarrays sanft und vertikal in drei Tröge mit mindestens 15 mL PBS abwaschen ungebundenen Zellen (20 s pro Waschgang). </li><li> Bereiten Sie 3,7% (w / v) Formaldehyd in PBS zu den Zellen und Antikörper durch Vernetzung zu beheben. Gently Pipette etwa 1 mL der Nitrozellulose Abschnitt des Microarray-Abdeckung. Inkubieren für 20 min bei Raumtemperatur. </li><li> Next dip-Microarrays in 3 Änderungen der PBS (15 ml, 30 s pro Stück) zum Auswaschen überschüssigen Formaldehyd. </li><li> Wischen Sie die Kanten-und Rückseite der Glasplatte mit Kimwipes und scannen Sie die Microarray mit dem DotScan Scanner Zeit wird die Nitrocellulose Abschnitt feucht. Die optische Abtastung bietet das Antigen Expressionsmuster einer gemischten Zellpopulation zB CRC-Zellen, Leukozyten und andere Stromazellen des Tumors. </li></ol> 4. Fluoreszenz-Multiplexing <ol><li> Entfernen Sie die Microarray vom Scanner und anwenden 200 ul Blocking-Puffer (2% w / v BSA, 2% hitzeinaktiviertem humanem AB-Serum, PBS, pH 7,3). Inkubieren es in der Microarray-Fach bei Raumtemperatur für 20 min. </li><li> Bereiten Sie die multiplexing Lösung in ein Eppendorf-Röhrchen mit Alufolie abgedeckt: 20 ul Phycoerythrin-anti-CD3 (Beckman Coulter, Gladesville, NSW, Australien, # IM12824; 1/7.5 Endverdünnung), 10 ul Alexa Fluor 647-anti-EpCAM (Biolegend, San Diego, CA, USA; 1 / 15 Verdünnung), 2 ul der hitzeinaktiviertem humanem AB-Serum (Sigma-Aldrich, Castle Hill, NSW, Australien) und 118 ul Blocking-Puffer. </li><li> Ablassen über Blockierungspuffer aus dem Mikroarray und Pipette das Multiplexing-Lösung auf die Nitrozellulose Abschnitt Verbreitung gleichmäßig. Inkubieren für 30 min im Dunkeln bei Raumtemperatur. </li><li> Tauchen Sie den Microarray vertikal in drei Mulden von 15 ml frischem PBS; (30 s pro Stück). </li><li> Lassen Sie die Microarrays trocken im Dunkeln und bei 4 ° C in einer Diashow angezeigt. Die Microarray kann in der Dunkelheit für bis zu 3 Monaten ohne Verlust der Fluoreszenz gespeichert. </li><li> Scan der trockenen Mikroarray mit einem Typhoon FLA 9000 Scanner (GE Healthcare, Rydalmere, NSW, Australien) mit einer Auflösung von 50 (532 nm Laser, 580 BP30 Emissionsfilter für PE. 633 nm-Laser und 670 BP30-Emission-Filter für Alexa 647). Die Mikroarrays werden mit der Nitrozellulose-Seite nach unten auf das Glas Scanner Fach gescannt. </li><li> Speichern Sie die fluoreszierende Bilder im TIFF-Dateien und mit Photoshop gesetzt Bildgröße 17 x 25 cm und eine Auflösung von 72 Pixel / cm. Importieren Sie die Grafik in das DotScan Analyse-Software, um die Intensität von Punkten zu analysieren. </li><li> Die DotReader fängt ein digitales Bild des Punktes Bindungsmuster und quantifiziert die Dichte der Zelle bindet jeder Antikörper Punkt auf einem 8-Bit Grau Skala (1-256 U). Gelegentliche unspezifische Isotyp-Kontroll-Bindung wurde die Bindung von Werten für Antikörper mit entsprechenden Immunglobulin-Isotypen abgezogen. Dot Fluoreszenzintensitäten für jeden Microarray wurden gegen den hellsten Punkt bei 100% Intensität normalisiert. Signal / spot Stärke wurde in einem. Xml-Datei (Rohdaten) aufgezeichnet oder dargestellt als Balkendiagramm in einer. Pdf-Datei (Abschlussbericht) </li><li> Microarray heatsmaps und hierarchische Clustering wurden mit MultiExperiment Viewer (MeV) Version 4.4 aus dem TM4 Microarray Software Suite ( <a href="http://www.tm4.org/mev.html">http://www.tm4.org/mev.html</a> ). Hierarchical Clustering wurde am Hintergrund bereinigte Daten mit MeV mit komplettem Linkage-Analyse durchgeführt. Euklidische Distanz wurde für Ähnlichkeitsmaß verwendet. Die 2-tailed Student-t-Test mit gleicher Varianz wurde verwendet, um die statistische Signifikanz der Ergebnisse zu bestimmen. </li></ol> 5. Repräsentative Ergebnisse: Ergebnisse aus dem DotScan Microarray soll zeigen konsistente Zelle Bindungsmuster zwischen doppelten Arrays. Starke Ausrichtung dot Bindung (CD44/CD29) ermöglicht ein Raster über das Array platziert werden. Abbildung 2 zeigt ein Beispiel für eine optimale Zell-Abscheidung und-Multiplexing. Abbildung 3 zeigt einige häufig auftretende Probleme während der Zellteilung erfassen und die möglichen Lösungen begegnet. Die Microarray-Zell-Bindung Ergebnisse können durch die Messung dot Intensitäten auf einem grauen Skala von 1 bis 256 ausgedrückt quantifiziert werden. Abbildung 4 zeigt numerische Daten aus 58 chirurgischen CRC-Proben, mit EpCAM-Alexa 647 Antikörper gefärbt, als Heatmap mit hierarchischen Clustering. Auch wenn die Anzahl der Proben ist beschränkt, meist CRCs der gleichen Bühne, um die Cluster in der gleichen Gruppe. <img src="/files/ftp_upload/3322/3322fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Zellbindung Muster der klinischen Darmkrebs Tumor (Australian Clinic-pathologische Staging, ACP Bühne B1). (A) DotScan Antikörper Schlüssel mit Standorten der Antikörper für die linke Hälfte der doppelten Microarray (umrissen). Der obere Abschnitt enthält den ursprünglichen 82-Antikörpern aus dem DotScan Leukämie-Mikroarray. Eine weitere 40-Antikörper, entsprechend spezifische Oberflächenantigene festgestellt, dass in der Literatur hoch reguliert, wurden als CRC &quot;Satelliten&quot; Microarray aufgenommen. Der untere Teil besteht aus Isotypkontrolle Antikörper (b) Optische Aufnahme CRC-Zellen die Bindung an den Microarray. (C) CD3 Abbildungsfunktion T-Zellen. (D) EpCAM Abbildungsfunktion CRC-Zellen. <img src="/files/ftp_upload/3322/3322fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Beispiele für schlechte DotScan Ergebnisse und mögliche Lösungen. (A) Niedrige Zellbindung; Lösung: Vergewissern Sie sich mindestens 4x106 lebensfähige Zellen sind auf dem Array (b) Isotypkontrolle verbindlich und unspezifische Zellbindung; Lösung: add hitzeinaktiviertem humanem AB-Serum zur Probe vor der Inkubation auf Mikroarray zu minimieren Isotypkontrolle bindend. Gelegentlich tritt eine kleine Menge an nicht-spezifische Bindung von Zellen an die Nitrozellulose nicht mit CRC-Proben und keinen signifikanten Einfluss auf die Ergebnisse. (C) Nitrocellulose Austrocknen während der Inkubation; Lösung: Sicherstellung der Probe deckt den gesamten Bereich Nitrocellulose und Microarray wird auf eine flache Oberfläche inkubiert. (D) High Hintergrund Artefakte; Lösung: Sicherstellung derMicroarray wird gründlich nach Inkubation gewaschen. <img src="/files/ftp_upload/3322/3322fig4.jpg" alt="Abbildung 4" /> Abbildung 4. DotScan Analyse-Software generierten Balkendiagramme darstellen Zellbindung Dichten auf einem grauen Skala von 1 bis 256. Die Zahlen auf der Achse beziehen sich auf CD-Antigene. Andere Abkürzungen werden TCR, T-Zell-Rezeptor, κ, λ, Immunglobulin leichten Ketten; sIg, Oberflächen-Immunglobulin, DCC, in der Darmkrebs-Protein gestrichen EGFR, epidermal growth factor receptor, FAP, Fibroblasten-Aktivierungs-Protein, HLA-A, B, C HLA-DR, humane Leukozyten-Antigene DR und A, B, C nicht überschreiten; MICA, MHC-Klasse-I-Kette-related protein A; MMP-14, Matrix Metallopeptidase 14; pIgR, polymere Immunglobulin Rezeptor; TSP-1, Thrombospondin-1; Mabthera, humanisierter Anti-CD20. <a href="http://www.jove.com/files/ftp_upload/3322/3322fig4large.jpg">Klicken Sie hier für eine größere Ansicht</a> .

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In diesem Video zeigen wir, wie das DotScan Antikörper Microarray in einer einfachen, semi-quantitative Weg, um Oberflächen-Antigen-Profile für Zellpopulationen aus CRC Gewebe Studie verwendet werden kann. Die Erteilung einer tragfähigen Einzel Zellsuspension aus Gewebe ist entscheidend für den Erfolg des Experiments, weil die Energie-abhängige Prozesse (zB, Antigen Begrenzung und / oder Pseudopodien-Bildung), für feste Bindung von ganzen Zellen zu Antikörper Punkte während der Inku...

The author's email has been corrected in the publication of <a href="http://www.jove.com/video/3322">Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing</a>. There was an error with the author, Jerry Zhou's, email. The author's email has been updated to:
j.zhou@uws.edu.au
from:
jzho7551@mail.usyd.edu.au

Erratum: Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

<table border="1"><tbody><tr><th> Name des Reagens oder Ausrüstung </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare </th></tr><tr><td> Hanks &#39;balanced salt solution </td><td> Sigma-Aldrich </td><td> H6136-10X1L </td><td> Buffered mit 25 mM Hepes (Sigma # H3375) </td></tr><tr><td> Airpure biologischen Sicherheitswerkbank der Klasse II </td><td> Westinghouse </td><td> 1687-2340/612 </td><td></td></tr><tr><td> Chirurgische Klingen </td><td> Livingstone </td><td> 090609 </td><td> Packung mit 100 Stück </td></tr><tr><td> RPMI 1640 mit 2 mM Hepes </td><td> Sigma-Aldrich </td><td> R4130-10X1L </td><td></td></tr><tr><td> Collagenase Typ 4 </td><td> Worthington </td><td> 4188 </td><td></td></tr><tr><td> Deoxyribonuclease 1 </td><td> Sigma-Aldrich </td><td> DN25-1G </td><td></td></tr><tr><td> Terumo Spritze (10 ml) </td><td> Terumo </td><td> MS +10 L </td><td> Box von 100 </td></tr><tr><td> Filcon Filter (200 um) </td><td> BD Biosciences </td><td> 340615 </td><td></td></tr><tr><td> Filcon Filter (50 um) </td><td> Filcon Filter (50 um) </td><td> Filcon Filter (50 um) Filcon Filter (50 um) 340603 </td><td></td></tr><tr><td> Fötalem Kälberserum </td><td> Gibco / Invitrogen </td><td> 10099-141 </td><td></td></tr><tr><td> Centrifuge 5810 R </td><td> Eppendorf </td><td> 7017 </td><td></td></tr><tr><td> Dimethylsulfoxid </td><td> Sigma-Aldrich </td><td> D2650 </td><td></td></tr><tr><td> Trypanblau </td><td> Sigma-Aldrich </td><td> T8154 </td><td></td></tr><tr><td> Hemocymeter Technocolor Neubar </td><td> Hirschmann </td><td> nicht verfügbar </td><td></td></tr><tr><td> Lichtmikroskop </td><td> Nikon </td><td> Nikon TMS </td><td></td></tr><tr><td> Cyrovial Rohre </td><td> Greiner Bio-One </td><td> 121278 </td><td></td></tr><tr><td> Cryo Einfrieren contrainer </td><td> Nalgene </td><td> 5100-0001 </td><td></td></tr><tr><td> DotScan Antikörper Microarray-Kit </td><td> Medsaic </td><td> nicht verfügbar </td><td></td></tr><tr><td> DotScan Microarray Waschküvette </td><td> Medsaic </td><td> nicht verfügbar </td><td></td></tr><tr><td> Kimwipes </td><td> Kimberly-Clark </td><td> 4103 </td><td></td></tr><tr><td> Formaldehyd 37% </td><td> Sigma-Aldrich </td><td> F1635-500ML </td><td></td></tr><tr><td> DotReaderTM </td><td> Medsaic </td><td> nicht verfügbar </td><td></td></tr><tr><td> Rinderserumalbumin </td><td> Sigma-Aldrich </td><td> A9418-10G </td><td></td></tr><tr><td> Heat-inaktivierten AB-Serum 2% </td><td> Invitrogen </td><td> 34005100 </td><td></td></tr><tr><td> Phycoerythrin-konjugierten CD3 </td><td> Beckman Coulter </td><td> ET386 </td><td></td></tr><tr><td> AlexaFluor647-konjugierten EpCAM </td><td> BioLegend </td><td> 324212 </td><td></td></tr><tr><td> Typhoon FLA 9000 </td><td> GE Healthcare </td><td> 28-9558-08 </td><td> 532 nm-Laser, 580 BP30 Emissionsfilter für PE. 633 nm-Laser und 670 BP30 Emissionsfilter für Alexa647 </td></tr><tr><td> MultiExperiment-Viewer v4.4 </td><td> TM4 Microarray Software Suite </td><td> Open - Source-Software (Ref 11) </td><td></td></tr></tbody></table>

colorectal cancer cell surface protein profiling using an antibody microarray and fluorescence multiplexing

Die aktuelle Prognose und Klassifikation von CRC basiert auf Staging-Systeme, die histopathologischen und klinischen Befunden zu integrieren. Doch in der Mehrzahl der CRC Fällen ist Zelldysfunktion das Ergebnis zahlreicher Mutationen, die Protein-Expression und posttranslationale Modifikation 1 ändern. Eine Reihe von Zell-Oberflächen-Antigene, einschließlich Cluster der Differenzierung (CD)-Antigene wurden als potenzielle prognostische oder metastasiertem Biomarker in CRC identifiziert worden. Diese Antigene bilden ideale Biomarker als ihren Ausdruck oft ändert sich mit Tumorprogression oder Wechselwirkungen mit anderen Zelltypen, wie zB Tumor-infiltrierenden Lymphozyten (TIL) und Tumor-assoziierte Makrophagen (TAM). Die Verwendung von Immunhistochemie (IHC) für Krebs sub-Klassifikation und Prognose ist gut für einige Tumorarten 2,3 etabliert. Es wurde jedoch keine einzige &quot;Marker&quot; prognostische Signifikanz gezeigt größer als klinisch-pathologischen Staging-oder gewonnen breite Akzeptanz für den Einsatz in der Routine-Pathologie Meldung aller CRC Fällen. Ein neuerer Ansatz, um prognostische Stratifizierung Krankheitsphänotypen setzt auf Oberflächenprotein Profile mit mehreren &quot;Marker&quot;. Während Expression Profiling von Tumoren mit Proteom-Techniken wie iTRAQ ist ein leistungsfähiges Werkzeug für die Entdeckung der biomarkers4, ist es nicht optimal für den routinemäßigen Einsatz in diagnostischen Laboratorien und kann nicht unterscheiden, verschiedene Zelltypen in eine gemischte Bevölkerung. Darüber hinaus werden große Mengen von Tumorgewebe für die Profilierung des gereinigten Plasmamembran Glykoproteine ​​durch diese Methoden erforderlich. In diesem Video haben wir beschrieben, eine einfache Methode zur Oberfläche Proteom-Profiling von lebensfähigen Zellen aus aufgeschlüsselte CRC-Proben mit einem DotScan CRC Antikörper Microarray. Die 122-Antikörper Microarray besteht aus einem Standard-82-Antikörper Region die Anerkennung einer Reihe von Lineage-spezifische Leukozyten-Marker, Adhäsionsmoleküle, Rezeptoren und Marker der Entzündung und Immunantwort 5, zusammen mit einem Satelliten-Region für den Nachweis von 40 potentiell prognostischer Marker für CRC . Die Zellen sind nur auf Antikörper, für die sie ausdrücken, das entsprechende Antigen erfasst. Die Zelldichte pro Punkt, durch optische Abtastung ermittelt, spiegelt der Anteil der Zellen, die das Antigen, das Niveau der Expression des Antigens und der Affinität des Antikörpers 6. Für CRC Gewebe oder normalen Darmschleimhaut, spiegeln optische Scans der Immunphänotyp von gemischten Populationen von Zellen. Fluoreszenz-Multiplexing können dann verwendet werden, um ausgewählte Sub-Populationen von Zellen von Interesse auf dem Array erfasst Profil. Zum Beispiel, Alexa 647-anti-Epithelial Cell Adhesion Molecule (EpCAM, CD326) ist ein pan-epitheliales Differenzierungsantigen, die zur CRC-Zellen und auch Epithelzellen der normalen Darmschleimhaut erkennen war, während Phycoerythrin-anti-CD3, wurde verwendet, zu erkennen infiltrierenden T-Zellen 7. Das DotScan CRC Microarray sollte der Prototyp für eine diagnostische Alternative zu den anatomisch-basierte CRC Staging-System sein.

Watch this Scientific Journal Video about Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing at JoVE.com

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

Wir beschrieben ein Verfahren für die Zerlegung von Darmkrebs (CRC), lebensfähige einzelne Zellen, die dann auf kundenspezifische Antikörper Microarrays erkennen Oberflächen-Antigene (DotScan CRC-Microarray) erfasst zu produzieren. Sub-Populationen von Zellen gebunden, die Microarray kann durch Fluoreszenz-Multiplexing mit monoklonalen Antikörpern mit Fluoreszenzfarbstoffen markiert profiliert werden.

Darmkrebs Zelloberflächenprotein Profiling mit einem Antikörper Microarray und Fluoreszenz Multiplexing

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority ...

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Research

JoVE Journal

Immunology and Infection

13.9K Aufrufe.  University of Sydney. Wir beschrieben ein Verfahren für die Zerlegung von Darmkrebs (CRC), lebensfähige einzelne Zellen, die dann auf kundenspezifische Antikörper Microarrays erkennen Oberflächen-Antigene (DotScan CRC-Microarray) erfasst zu produzieren. Sub-Populationen von Zellen gebunden, die Microarray kann durch Fluoreszenz-Multiplexing mit monoklonalen Antikörpern mit Fluoreszenzfarbstoffen markiert profiliert werden.

Serie: Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

Mit einem Pan-Viral Microarray-Assay (Virochip) zur klinischen Proben, die für virale Erreger Bildschirm

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Forschung

Immunologie und Infektion

Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular localization can provide insights into the functional role of bacterial proteins. Surface localization of bacterial proteins is a key step towards identification of virulence factors involved in mechanisms of pathogenicity. 
Methods for fractionating leptospiral membranes 2-5 may be selective for a certain class of outer-membrane proteins (OMPs), such as lipoproteins vs. transmembrane OMPs, and therefore lead to misclassification. This likely is due to structural differences and how they are associated to the outer membrane. Lipoproteins are associated with membranes via a hydrophobic interaction between the N-terminal lipid moiety (three fatty acids) and the lipid bilayer phospholipids 6, 7. In contrast, transmembrane OMPs are typically integrated into the lipid bilayer by amphipathic &beta;-sheets arranged in a barrel-like structure 8, 9. In addition, presence of a protein in the outer-membrane does not necessarily guarantee that the protein or its domains are exposed on the surface. Spirochetal outer membranes are known to be fragile and therefore necessitate methods involving gentle manipulation of cells and inclusion of sub-surface protein controls to assess the integrity of the outer membrane.
Here, we present an immunofluorescence assay (IFA) method to directly assess surface exposure of proteins on intact leptospires. This method is based on recognition of leptospiral surface proteins by antigen-specific antibodies. Herein, antibodies specific for OmpL5410 are detetcted aftero binding to native, surface exposed epitopes. Comparison of antibody reactivity to intact versus permeabilized cells enables evaluation of cellular distribution and whether or not a protein is selectively present on leptospiral surface. The integrity of outer membrane should be assessed using antibody to one or more subsurface proteins, preferably located in the periplasm.
The surface IFA method can be used to analyze surface exposure of any leptospiral protein to which specific antibodies are available. Both the usefulness and limitation of the method depends on whether the antibodies employed are able to bind to native epitopes. Since antibodies often are raised against recombinant proteins, epitopes of native, surface-exposed proteins may not be recognized. Nevertheless, the surface IFA method is a valuable tool for studying components of intact bacterial surfaces. This method can be applied not only for leptospires but also other spirochetes and gram-negative bacteria. For stronger conclusions regarding surface-exposure of OMPs, a comprehensive approach involving several cell localization methods is recommended 10.

An efficient method to assess surface-exposure of leptospiral proteins is described. The method is specifically designed to avoid disruption of the fragile outer membrane of leptospiral cells. This technique requires employment of several negative controls to assess the integrity of the outer membrane and specificity of antibody reaction.

Immuno-Fluoreszenz-Assay von Leptospira-Surface-exponierten Proteine

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

Einzel-Zell-Analyse Bacillus subtilis Biofilme mittels Fluoreszenzmikroskopie und Durchflusszytometrie

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

Visualisierung der bakteriellen Toxin induzierten Reaktionen mit Live Cell Fluoreszenzmikroskopie

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

Komparativen Ansatz, um die Landschaft von Wirt-Pathogen-Protein-Protein-Wechselwirkungen charakterisieren

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

Ein miniaturisiertes Glycan Microarray-Test zur Beurteilung der Avidität und Spezifität von Influenza-A-Virus Hämagglutininarten

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, immunodiagnostics and more recently as therapeutic agents. Traditional methods for labeling antibodies with small molecules require purified antibodies at relatively high concentration, involve multiple dialysis steps and have limited throughput. However, several applications, including the field of Antibody Drug Conjugates (ADCs), will benefit from new methods that will allow labeling of antibodies directly from cell media. Such methods may allow antibodies to be screened in biologically relevant assays, for example, the receptor-mediated antibody internalization assay in the case of ADCs. Here, we describe a method (on-bead method) that enables labeling of small amounts of antibodies directly from cell media. This approach utilizes high capacity magnetic Protein A and Protein G affinity beads to capture antibodies from the cell media followed by labeling with small molecules using either amine or thiol chemistry and subsequent elution of the labeled antibodies. Taking fluorescent dyes as surrogates for small molecules, we demonstrate the on-bead labeling of three different mouse antibodies directly from cell media using both amine and thiol labeling chemistry. The high binding affinity of antibodies to Protein A and Protein G ensures high recoveries as well as high purity of the labeled antibodies. In addition, use of magnetic beads allows multiple samples to be handled manually, thereby significantly improving labeling throughput.

The on-bead method for labeling antibodies with small molecules enables labeling of a small amount of antibodies directly from cell media. This method is compatible with amine and thiol chemistry, and can handle multiple samples in parallel, manually or using automated platforms.

Antikörpermarkierung mit Fluoreszenzfarbstoffen unter Verwendung von magnetischen Protein A und Protein G-Kügelchen

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

Ein Protein-Microarray-Assay für die serologische Bestimmung der Antigen-spezifische Antikörper Antworten folgende Clostridium Difficile Infektion

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

Einzellige Quantifizierung der mRNA und Oberfläche Protein-Expression in Simian Immunodeficiency Virus-infizierten CD4+ T Zellen isoliert von Rhesus-Makaken

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.