Nous remercions le personnel des laboratoires de pathologie anatomique du Royal Prince Alfred et les hôpitaux rapatriement Concord pour la collecte des échantillons frais du CRC et de la muqueuse intestinale normale. Le travail a été financé par une subvention Cancer Institute Nouvelle-Galles du Sud Programme translationnelle.

<img src="/files/ftp_upload/3322/3322fig1.jpg" alt="Figure 1" /> Le flux de travail figure 1. Pour la préparation d&#39;une suspension de cellules vivantes à partir d&#39;un échantillon de chirurgie de la CRC. 1. Désagrégation de l&#39;échantillon clinique Tous les échantillons ont été prélevés dans l&#39;hôpital Royal Prince Alfred (Camperdown, NSW, Australie) et l&#39;Hôpital de rapatriement Concord (Concord West, NSW, Australie) avec le consentement éclairé du Protocole n ° X08-164. <ol><li> Recueillir frais cancer colorectal (CCR) ou adénome spécimens, et la muqueuse intestinale normale d&#39;au moins 10 cm de la tumeur. Conserver les échantillons au pH du sel de Hank solution équilibrée 7.3 (HBSS) à 4 ° C pendant 12 h après la résection. </li><li> Suivez les règles de sécurité pour les agents pathogènes humains, traiter tous les échantillons cliniques dans un coffret de sécurité biologique de classe II. Disséquer les échantillons en cubes de 2 mm dans une boîte de Pétri en utilisant deux lames de bistouri. </li><li> Incuber la tumeur et des tissus normaux dans des tubes Eppendorf séparée avec occasionnellement un mélange doux pendant 60 min à 37 ° C avec un volume égal de milieu RPMI 1640 contenant 2% (v / v) la collagénase de type 4 (Worthington, Lakewood, New Jersey, USA) et 0,1 désoxyribonucléase% (p / v) J&#39;ai du pancréas bovin (DNAse I; Sigma-Aldrich). </li><li> Groupe semi-digérée tissus à travers une passoire fine en utilisant un fil de piston de la seringue de 10 ml, les cellules se laver travers avec HBSS. </li><li> Passe résultant suspension cellulaire à 200 um et 50 um filtres Filcon (BD Biosciences) pour enlever les agrégats de cellules. La plupart des agrégats d&#39;ADN, de mucus et de cellules sont supprimés dans cette série de filtrations. </li><li> Centrifugeuse suspensions cellulaires à 400 xg à 20 ° pendant 5 min. </li><li> Culots cellulaires Remettre en suspension dans la chaleur FCS inactivé, contenant 10% de diméthylsulfoxyde (DMSO), de geler lentement dans cryotubes et conserver à -80 °. Le processus de congélation tend à réduire de mucus dans l&#39;échantillon et lyse des globules rouges. </li></ol> 2. La préparation des échantillons pour la capture de la cellule <ol><li> Dégeler rapidement des échantillons dans un bain d&#39;eau à 37 ° et remettre les cellules dans 10 ml de HBSS pour laver le DMSO. </li><li> Centrifugeuse suspensions cellulaires à 410 xg à 20 ° pendant 5 min. </li><li> Décanter le surnageant et remettre le culot cellulaire dans 500 ul de HBSS. </li><li> Traiter l&#39;échantillon avec 0,1% (p / v) DNAse I pendant 20 min à température ambiante. </li><li> Mélanger 10 uL de chaque suspension cellulaire avec un volume égal de bleu de trypan et charger 10 uL du mélange dans un hématimètre. En utilisant un microscope optique à 100 fois le grossissement, le comptage de cellules viables, qui apparaissent clairement en raison d&#39;exclusion du bleu trypan, tandis que les cellules mortes prennent le colorant. Un minimum de 4 x 10 6 cellules viables est requise pour la capture de cellules sur la puce. </li><li> Après traitement à la DNase, remettre en suspension les cellules en suspension dans 10 ml de HBSS et centrifuger à 410 xg à 20 ° pendant 5 min. </li><li> Décanter les surnageants et les culots cellulaires remettre en suspension dans du RPMI 1640 à un volume final de 200 pl. </li></ol> 3. Anticorps de capture cellulaire biopuces <ol><li> Humidifiez les biopuces d&#39;anticorps DotScan en plongeant dans la section de nitrocellulose tampon phosphate salin (PBS) pendant environ 20 s. Essuyer soigneusement les bords de verre de la puce à ADN avec Kimwipes plié, en évitant de toucher la section de nitrocellulose. </li><li> Ajouter de l&#39;eau dans le bac d&#39;incubation de microréseaux pour offrir une chambre humide. Placer la puce dans la chambre et la pipette de la suspension cellulaire dans du RPMI 1640 sur la section de nitrocellulose humide. Pipeter gouttes sur chaque coin de la nitrocellulose afin d&#39;assurer une répartition homogène de cellules. </li><li> Incuber les puces à 37 ° C pendant 1 h. L&#39;incubation des cellules permet de s&#39;installer et entrer en contact avec des anticorps sur la puce. Les cellules exprimant les antigènes de surface correspondant à l&#39;anticorps ils débarquent sur sera capturée. </li><li> Après incubation, tremper les microarrays doucement et verticalement en trois bacs contenant au moins 15 ml de PBS à laver les cellules non liée (20 s par lavage). </li><li> Préparer 3,7% (p / v) de formaldéhyde dans du PBS pour fixer les cellules et d&#39;anticorps par réticulation. Délicatement la pipette environ 1 mL pour couvrir la section de nitrocellulose du microréseau. Incuber pendant 20 min à température ambiante. </li><li> Suivant DIP puces en 3 changements de PBS (15 mL; 30 s chacun) pour laver le formaldéhyde en excès. </li><li> Essuyez les bords et le dos de la lame de verre avec Kimwipes et scanner la puce à l&#39;aide du scanner de tout DotScan, la section de nitrocellulose est humide. Le balayage optique fournit le modèle de l&#39;expression des antigènes d&#39;une population de cellules cellules mixtes CRC par exemple, les leucocytes et autres cellules du stroma de la tumeur. </li></ol> 4. Fluorescence multiplexage <ol><li> Retirer la puce à partir du scanner et d&#39;appliquer 200 uL de tampon de blocage (2% p / v de BSA, 2% inactivé à la chaleur sérum humain AB, PBS, pH 7,3). Il Incuber dans le plateau de biopuces à température ambiante pendant 20 min. </li><li> Préparer la multiparelexing solution dans un tube Eppendorf recouverts d&#39;une feuille d&#39;aluminium: 20 pl phycoérythrine anti-CD3 (Beckman Coulter, Gladesville, NSW, Australie, # IM12824; 1/7.5 dilution finale), 10 uL Alexa Fluor 647-anti-EpCAM (Biolegend, San Diego, Californie, Etats-Unis; 1 / 15 de dilution), 2 pl de la chaleur sérum humain inactivé AB (Sigma-Aldrich, Castle Hill, NSW, Australie) et 118 uL de tampon de blocage. </li><li> Égoutter l&#39;excès tampon de blocage de la puce et la pipette la solution de multiplexage sur la section de nitrocellulose, répandre uniformément. Incuber pendant 30 min dans le noir à température ambiante. </li><li> Tremper les biopuces verticalement en trois bacs de 15 ml de PBS frais, (30 s chacun). </li><li> Laissez les microarrays sécher dans l&#39;obscurité et stocker à 4 ° C dans une boîte à lames. Les puces à ADN peuvent être stockés dans l&#39;obscurité pendant jusqu&#39;à 3 mois sans perte de fluorescence. </li><li> Numérisez les biopuces à sec en utilisant un typhon FLA 9000 Scanner (GE Healthcare, Rydalmere, NSW, Australie) avec la résolution fixée à 50 (532 nm laser, 580, BP30 émission pour les PE filtre. Laser 633 nm et 670 BP30 filtre d&#39;émission pour Alexa 647). Les biopuces sont scannées avec le côté orienté vers le bas de nitrocellulose sur le plateau de la vitre du scanner. </li><li> Enregistrer les images fluorescentes sous forme de fichiers TIFF et Photoshop en utilisant la taille d&#39;image réglée sur 17 x 25 cm et de résolution de 72 pixels / cm. Importer l&#39;image dans le logiciel d&#39;analyse DotScan d&#39;analyser l&#39;intensité de points. </li><li> Le DotReader capture une image numérique de la configuration de points de liaison et quantifie la densité de la liaison cellulaire sur chaque point d&#39;anticorps sur une échelle grisaille 8 bits (1-256 U). Occasional liaison non spécifique contrôle isotypique a été soustraite de valeurs contraignantes pour les anticorps avec isotypes d&#39;immunoglobulines correspondantes. Dot intensités de fluorescence pour chaque puce ont été normalisés par rapport à la plus brillante de points fixé à 100% d&#39;intensité. Signal / spot force a été enregistré dans un fichier. XML (données brutes), ou représenté comme un graphique à barres dans un fichier. Pdf (rapport final) </li><li> Microarray heatsmaps et le clustering hiérarchique ont été effectuées en utilisant MultiExperiment Viewer (MeV) la version 4.4 de la suite logicielle TM4 Microarray ( <a href="http://www.tm4.org/mev.html">http://www.tm4.org/mev.html</a> ). Classification hiérarchique a été réalisée sur des données de base ajustée à l&#39;aide MeV avec l&#39;analyse de liaison complète. Distance euclidienne a été utilisée pour mesure de similarité. L&#39;élève 2-tailed test t de variance égale a été utilisée pour déterminer la signification statistique des résultats. </li></ol> 5. Les résultats représentatifs: Résultats de la biopuce DotScan devrait montrer les modèles cellulaires compatibles contraignante entre les tableaux en double. Forte d&#39;alignement de points de liaison (CD44/CD29) permet une grille pour être placé sur la zone de tableau. La figure 2 montre un exemple de capture cellulaire optimale et le multiplexage. La figure 3 montre certains problèmes courants rencontrés lors de la capture cellulaire et les solutions possibles. Les résultats de cellules biopuces liaison peut être quantifiée en mesurant l&#39;intensité de points sur une échelle grisaille allant de 1 à 256. La figure 4 montre les données numériques à partir de 58 échantillons CRC chirurgicale, colorées avec EpCAM-Alexa 647 anticorps, comme un heatmap avec classification hiérarchique. Même si le nombre d&#39;échantillons est limitée, CRC d&#39;un même étage ont tendance à se regrouper dans un même groupe. <img src="/files/ftp_upload/3322/3322fig2.jpg" alt="Figure 2" /> Figure 2. Motif de liaison des cellules tumorales du cancer colorectal cliniques (Australian Clinique-pathologique Staging, ACP étape B1). (A) d&#39;anticorps DotScan clés indiquant les emplacements des anticorps pour la moitié gauche de la puce en double (décrites). La partie supérieure contient les 82 anticorps d&#39;origine du microréseau leucémies DotScan. 40 autres anticorps, correspondant à des antigènes de surface spécifiques jugés sur-régulée dans la littérature, ont été ajoutés comme biopuces «satellite» d&#39;un CRC. La section inférieure se compose d&#39;anticorps isotype contrôle (b) d&#39;image optique de cellules CRC se liant à la puce. (C) image de fluorescence montrant CD3 T-cellules. (D) EpCAM de fluorescence d&#39;image montrant des cellules CRC. <img src="/files/ftp_upload/3322/3322fig3.jpg" alt="Figure 3" /> Figure 3. Exemples de mauvais résultats DotScan et les solutions possibles. (A) de cellules à faible liaison; Solution: Assurez au moins 4x106 cellules viables sont sur le tableau (b) la cellule de contrôle isotypique contraignants et non contraignants spécifiques; solution: ajouter de la chaleur inactivé sérum humain AB d&#39;échantillon avant incubation sur microréseaux afin de minimiser isotype contrôle contraignant. Parfois, une petite quantité de liaison non spécifique des cellules de la nitrocellulose se produit avec des échantillons de CRC et n&#39;affecte pas significativement les résultats. (C) La nitrocellulose dessèchement pendant l&#39;incubation; solution: assurer échantillon couvre la section entière et de nitrocellulose est incubée biopuces sur une surface plane. (D) les artefacts de fond élevé; solution: assurer lamicroréseau est soigneusement lavé après incubation. <img src="/files/ftp_upload/3322/3322fig4.jpg" alt="Figure 4" /> Figure 4. Logiciel d&#39;analyse DotScan généré des graphiques à barres représentant des densités cellulaires contraignant sur ​​une échelle de gris allant de 1 à 256. Numéros sur l&#39;axe se référer à des antigènes CD. D&#39;autres abréviations sont TCR, T cell receptor; κ, λ, chaînes légères d&#39;immunoglobulines, sig, immunoglobulines de surface; CDC, supprimé en protéines du cancer colorectal; EGFR, récepteur épidermique de facteur de croissance; FAP, activation de la protéine fibroblastique; HLA-A, B, C HLA-DR, les antigènes de leucocytes humains DR et A, B, C respectivement; MICA, CMH de classe I liés à la chaîne de la protéine A; MMP-14, métallopeptidase matrice 14; plgR, récepteur des immunoglobulines polymériques; TSP-1, la thrombospondine-1; Mabthera, humanisé anti-CD20. <a href="http://www.jove.com/files/ftp_upload/3322/3322fig4large.jpg">Cliquez ici pour agrandir l&#39;image</a> .

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Dans cette vidéo, nous démontrons comment les biopuces d&#39;anticorps DotScan peut être utilisé dans un simple, semi-quantitative façon d&#39;étudier les profils d&#39;antigène de surface pour les populations de cellules du tissu CRC. L&#39;obtention d&#39;une suspension de cellules viables unique à partir de tissus est essentielle à la réussite de l&#39;expérience, parce que dépendant de l&#39;énergie des processus (par exemple, l&#39;antigène le recouvrement et / ou la forma...

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> Nom de réactif ou de l&#39;équipement </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires </th></tr><tr><td> Hanks solution salée équilibrée </td><td> Sigma-Aldrich </td><td> H6136-10X1L </td><td> Tamponné avec 25 mM HEPES (Sigma # H3375) </td></tr><tr><td> Airpure sécurité biologique de classe II du cabinet </td><td> Westinghouse </td><td> 1687-2340/612 </td><td></td></tr><tr><td> Lames chirurgicales </td><td> Livingstone </td><td> 090609 </td><td> Paquet de 100 </td></tr><tr><td> RPMI 1640 avec 2 mM Hepes </td><td> Sigma-Aldrich </td><td> R4130-10X1L </td><td></td></tr><tr><td> Collagénase de type 4 </td><td> Worthington </td><td> 4188 </td><td></td></tr><tr><td> Désoxyribonucléase 1 </td><td> Sigma-Aldrich </td><td> DN25-1G </td><td></td></tr><tr><td> Terumo Seringue (10 ml) </td><td> Terumo </td><td> SS 10 L </td><td> Boîte de 100 </td></tr><tr><td> Filcon filtre (200 m) </td><td> BD Biosciences </td><td> 340615 </td><td></td></tr><tr><td> Filcon filtre (50 microns) </td><td> Filcon filtre (50 microns) </td><td> Filcon filtre (50 microns) Filcon filtre (50 microns) 340 603 </td><td></td></tr><tr><td> Sérum de veau fœtal </td><td> Gibco / Invitrogen </td><td> 10099-141 </td><td></td></tr><tr><td> Centrifugeuse 5810 R </td><td> Eppendorf </td><td> 7017 </td><td></td></tr><tr><td> Diméthylsulfoxyde </td><td> Sigma-Aldrich </td><td> D2650 </td><td></td></tr><tr><td> Le bleu trypan </td><td> Sigma-Aldrich </td><td> T8154 </td><td></td></tr><tr><td> Hemocymeter Technocolor Neubar </td><td> Hirschmann </td><td> pas disponible </td><td></td></tr><tr><td> Microscope à lumière </td><td> Nikon </td><td> Nikon TMS </td><td></td></tr><tr><td> Tubes Cyrovial </td><td> Greiner Bio-One </td><td> 121278 </td><td></td></tr><tr><td> Contrainer congélation Cryo </td><td> Nalgene </td><td> 5100-0001 </td><td></td></tr><tr><td> DotScan anticorps microarray kit </td><td> Medsaic </td><td> pas disponible </td><td></td></tr><tr><td> Bac DotScan laver les biopuces </td><td> Medsaic </td><td> pas disponible </td><td></td></tr><tr><td> Kimwipes </td><td> Kimberly-Clark </td><td> 4103 </td><td></td></tr><tr><td> Formaldéhyde 37% </td><td> Sigma-Aldrich </td><td> F1635-500ML </td><td></td></tr><tr><td> DotReaderTM </td><td> Medsaic </td><td> pas disponible </td><td></td></tr><tr><td> L&#39;albumine sérique bovine </td><td> Sigma-Aldrich </td><td> A9418-10G </td><td></td></tr><tr><td> Inactivés par la chaleur du sérum AB 2% </td><td> Invitrogen </td><td> 34005100 </td><td></td></tr><tr><td> Phycoérythrine conjuguée CD3 </td><td> Beckman Coulter </td><td> ET386 </td><td></td></tr><tr><td> AlexaFluor647 conjugué EpCAM </td><td> BioLegend </td><td> 324212 </td><td></td></tr><tr><td> Typhon FLA 9000 </td><td> GE Healthcare </td><td> 28-9558-08 </td><td> 532 nm laser, 580, BP30 émission pour les PE filtre. 633 nm laser et 670 BP30 émissions pour Alexa647 filtre </td></tr><tr><td> MultiExperiment Viewer v4.4 </td><td> TM4 Microarray Suite logicielle </td><td> Open - source (Réf 11) </td><td></td></tr></tbody></table>

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

Le pronostic actuel et la classification des CRC repose sur la stadification des systèmes qui intègrent les résultats histopathologiques et cliniques. Cependant, dans la majorité des cas de CCR, le dysfonctionnement cellulaire est le résultat de nombreuses mutations qui modifient l&#39;expression des protéines et des modifications post-traductionnelles 1. Un certain nombre d&#39;antigènes de surface cellulaire, y compris les clusters de différenciation (CD) antigènes, ont été identifiés comme pronostiques potentiels ou métastatique chez les biomarqueurs CRC. Ces antigènes font biomarqueurs idéal comme leur expression change souvent avec la progression tumorale ou des interactions avec d&#39;autres types cellulaires, comme les lymphocytes infiltrant les tumeurs (TIL) et les macrophages associés aux tumeurs (TAM). L&#39;utilisation de l&#39;immunohistochimie (IHC) pour le cancer de la sous-classification et le pronostic est bien établi pour certains types de tumeurs 2,3. Cependant, pas un seul «marqueur» a montré l&#39;importance pronostique supérieure clinico-pathologiques ou de mise en scène gagné l&#39;acceptation large pour une utilisation dans les rapports de pathologie de routine de tous les cas, le CRC. Une approche plus récente à la stratification pronostique des phénotypes de la maladie repose sur les profils protéiques de surface en utilisant de multiples «marqueurs». Alors que le profil d&#39;expression des tumeurs en utilisant des techniques telles que la protéomique iTRAQ est un outil puissant pour la découverte de biomarkers4, il n&#39;est pas optimale pour une utilisation de routine dans les laboratoires de diagnostic et ne peuvent pas distinguer différents types de cellules dans une population mixte. En outre, de grandes quantités de tissus tumoraux sont nécessaires pour le profilage des glycoprotéines purifiées membrane plasmique par ces méthodes. Dans cette vidéo, nous avons décrit une méthode simple pour le profilage de la surface du protéome de cellules viables à partir d&#39;échantillons CRC ventilées en utilisant une puce à anticorps DotScan CRC. La puce 122-anticorps se compose d&#39;une norme 82-anticorps reconnaissant la région une gamme de la lignée des marqueurs spécifiques des leucocytes, des molécules d&#39;adhésion, les récepteurs et les marqueurs de l&#39;inflammation et la réponse immunitaire 5, avec une région par satellite pour la détection de 40 marqueurs potentiellement pronostique pour le CRC . Les cellules sont capturées uniquement sur les anticorps pour lequel ils expriment l&#39;antigène correspondant. La densité cellulaire par point, déterminé par balayage optique, reflète la proportion de cellules exprimant l&#39;antigène, le niveau d&#39;expression de l&#39;antigène et l&#39;affinité de l&#39;anticorps 6. Pour les tissus normaux CRC ou la muqueuse intestinale, balayages optiques reflètent l&#39;immunophénotype des populations mixtes de cellules. Multiplexage par fluorescence peut alors être utilisé pour le profil sélectionné sous-populations de cellules d&#39;intérêt capturés sur le tableau. Par exemple, Alexa 647 molécules anti-adhérence des cellules épithéliales (EpCAM; CD326), est un antigène de différenciation pan-épithéliales qui a été utilisé pour détecter les cellules CRC et aussi les cellules épithéliales de la muqueuse intestinale normale, tandis que la phycoérythrine-anticorps anti-CD3, a été utilisé pour détecter l&#39;infiltration de cellules T 7. Le CRC DotScan biopuces devrait être le prototype d&#39;une alternative de diagnostic pour le système anatomique basée sur scène CRC.

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

Nous avons décrit une procédure pour la désagrégation du cancer colorectal (CCR) pour produire des cellules viables unique, qui sont ensuite saisies sur biopuces d&#39;anticorps reconnaissant les antigènes de surface sur mesure (CRC DotScan micropuce). Les sous-populations de cellules lié à la puce peut être profilée par multiplexage de fluorescence à l&#39;aide d&#39;anticorps monoclonaux marqués avec des colorants fluorescents.

Colorectal Cancer Cell Surface Protein profilage utilisant une biopuces d&#39;anticorps et de multiplexage par fluorescence

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

colorectal-cancer-cell-surface-protein-profiling-using-an-antibody

Research

JoVE Journal

Immunology and Infection

13.9K vues.  University of Sydney. Nous avons décrit une procédure pour la désagrégation du cancer colorectal (CCR) pour produire des cellules viables unique, qui sont ensuite saisies sur biopuces d'anticorps reconnaissant les antigènes de surface sur mesure (CRC DotScan micropuce). Les sous-populations de cellules lié à la puce peut être profilée par multiplexage de fluorescence à l'aide d'anticorps monoclonaux marqués avec des colorants fluorescents.

Vidéo: 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.

En utilisant un dosage Microarray pan-virale (Virochip) pour cribler des échantillons cliniques pour les pathogènes viraux

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 ...

Recherche

Immunologie et infection

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-fluorescence Dosage des leptospires exposées à la surface Protéines

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.

Analyse unicellulaire des<emBacillus subtilis> Les biofilms par microscopie à fluorescence et cytométrie de flux

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.

Visualisation des réponses toxine bactérienne induite en utilisant direct microscopie de fluorescence cellulaire

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.

Approche comparative caractérisent le paysage des interactions hôte-pathogène protéine-protéine

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.

A miniaturisé Glycan Microarray Assay pour l&#39;évaluation Avidité et Spécificité du virus grippal A Hémagglutinines

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.

Anticorps marquage avec des colorants fluorescents en utilisant une protéine magnétique A et protéine G Perles

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.

Une analyse de Microarray de protéine pour détermination sérologique de l’Infection à Clostridium difficile anticorps réponses suivantes spécifiques à l’antigène

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.

Unicellulaire quantification des ARNm et Expression de protéines de Surface CD4 infectées par le Virus d’immunodéficience simien+ T les cellules isolées de macaques rhésus

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.