Die Autoren bedanken sich bei allen EIB-Mitglieder für die Entwicklung dieser Technik zu danken. Diese Arbeit wurde zum Teil durch Grant-in-Aid for Scientific Research auf Priority Areas &quot;Membrane Traffic&quot; (HO) und &quot;Matrix of Infectious Phenomena&quot; (KH), Young Scientists (SF und KH) und Wissenschaftliche Forschung (HO unterstützt ) und Scientific Research on Innovative Areas &quot;Intrazelluläre Logistik&quot; (HO), vom Ministerium für Bildung, Kultur, Sport, Wissenschaft und Technologie in Japan.

1. Vorbereitung von Bakterienzellen <ol><li> Streak Glycerin gefüllt fluoreszierende Bakterien (z. B. GFP-exprimierenden S. typhimurium) auf LB-Agar-Platte mit 100 ug / ml Ampicillin. </li><li> Kultur einer einzelnen Kolonie von LB-Agar über Nacht in 2 ml der neuen LB-Medium. </li><li> 0,5 ml Bakterienkultur zu 4,5 ml der neuen LB-Medium und Inkubation bis optischen Dichte von 1,0 bei 600 nm erreicht ist. </li><li> Ernte Bakterienzellen durch Zentrifugation (3.000 xg, 5 min, 4 ° C). </li><li> Überstand verwerfen und waschen zweimal mit 5,0 ml sterilem Phosphatpuffer (PBS). </li><li> Resuspendieren Bakterienpellet mit 5 ml PBS, und verwenden Sie 50 ul der Suspension, die etwa 10 7 koloniebildenden Einheit (KBE) als Inokulum. </li><li> Bei Verwendung von fluoreszenzmarkierten Bakterien wurden die Bakterienzellen durch Fluoreszenzmarkierung Reagenz nach Standard-Protokoll bezeichnet. </li></ol> 2. Anesthesia <ol><li> Füllen Sie eine kleine Kunststoff-Box (10 x 10 x 5 cm) mit 5% (v / v) verdampft Isofluran mit Luft gemischt (Flussrate: 200 ml / min). </li><li> Anesthetize acht bis sechzehn schwachen alten männlichen oder weiblichen Mäusen in der Box. </li><li> Bewegen Sie die Maus zu einem Seziertisch nach der Narkose. </li><li> Kontinuierlich betäuben die Mäuse um 2% (v / v) verdampft Isofluran mit Luft gemischt (Flussrate: 200 ml / min) (Abbildung 1). Dies ist ein Terminal-Verfahren. </li></ol> 3. Ligiert Peyer-Patch Loop-Test <ol><li> Incise 1cm der Bauchhaut und dann schneiden Sie die Bauch-Peritoneum einer narkotisierten Maus und nehmen Sie den Dünndarm mit der Peyer-Patch. </li><li> Ligieren des Darms mit Nähgarn, kümmert sich um Blutgefäße zu vermeiden. Hinweis: nur binden einen Seite des Darmes, und lassen Sie die andere Seite zu verlieren. </li><li> Inject 50 ul Bakteriensuspension oder PBS (Kontrolle) mit einer Spritze in ligiert Peyer-Patch-Schleife auf dem losen side des Darms (Abbildung 2). </li><li> Bind Losseite, und schließen Sie die Maus den Bauch mit einem Clip. </li><li> Nach 1 h, entfernen Sie die ligiert Peyer-Patch-Schleife, und einschläfern mit der Maus durch Genickbruch. </li><li> Excise Peyer-Patch von ligiert Peyer-Patch-Schleife. </li><li> Flashing der apikalen Seite der Peyer-Patch von 1 ml PBS mit der Verwendung von Spritzen an einer Nadel, um überschüssige Schleimhaut Flüssigkeit und Bakterien zu entfernen. Flashen der Peyer-Patch weitere zwei Male. </li></ol> 4. Whole mount-Färbung und konfokale mikroskopische Analyse der Peyer-Patch <ol><li> Fix Peyer-Patch in BD Cytofix / Cytoperm Lösung auf Eis für 1 Stunde. </li><li> Wash Peyer-Patch dreimal mit 1 ml BD Perm / Wash-Puffer für 5 min und dann Block mit 1 ml Blocking-Puffer mit 0,1% (w / v) Saponin, 0,2% (w / v) BSA in PBS für 30 min auf Eis. </li><li> Add 200-fach verdünnt Anti-Maus-GP2 monoklonaler Antikörper (5 pg / ml) auf die Probe zu erkennenM-Zellen. </li><li> Inkubieren für 2 Stunden bei Raumtemperatur oder über Nacht bei 4 ° C. </li><li> Wash dreimal mit 1 ml kaltem PBS, fügen Sie dann 200-fach verdünnt Anti-Ratte-IgG-Antikörper, konjugiert mit DyLight549 (20 ug / ml) auf die Probe. </li><li> Inkubieren Sie die Probe auf Eis für 2 Stunden. </li><li> Wash dreimal mit 1 ml kaltem PBS, dann fügen Sie 50-fach verdünnt Alexa 633 konjugierten Phalloidin auf die Probe zu F-Aktin zu erkennen. </li><li> Inkubieren auf Eis für 2 Stunden. </li><li> Wash leicht mit 1 ml kaltem PBS. Dann legen Sie drei Minuten vor vier Stücke runden Deckglas auf einem Objektträger, und betten Sie die Proben in eine 30% ige Glycerin-Lösung in PBS auf der Folie (Abbildung 3). </li><li> Beachten Proben mit einer DM-IRE2 konfokalen Laser Scanning Mikroskop oder einem DeltaVision Restoration Dekonvolution Mikroskop. </li></ol> 5. Repräsentative Ergebnisse: Die Probe beispielsweise einer Maus ligiert Peyer-Patch Loop-Test mit GFP-S. Typhimurium wurde mit einem DeltaVision Restoration Dekonvolution Mikroskop (Abbildung 4) beobachtet. GFP-S. typhimurium wird durch GP2 + M-Zellen transcytosed. Eine Maus Peyer-Patch Darmschlinge Assay identifizieren kann die Art der Kommensalen oder pathogene Bakterien, die durch M-Zellen transcytosed werden kann. <img alt="Abbildung 1" src="/files/ftp_upload/3225/3225fig1.jpg" /> Abbildung 1. Anesthesia von Mäusen unter verdampft Isofluran Zustand. Verdampft Isofluran wurde von einer Anästhesie-dedicated Gerät (links) geliefert. <img alt="Abbildung 2" src="/files/ftp_upload/3225/3225fig2.jpg" /> Abbildung 2. Zusammenfassung der ligiert Peyer-Patch Darmschlinge Assay. Die fluoreszierenden Bakteriensuspension wurde mit einer Spritze in ligiert Peyer&#39;schen Patch Schleife auf der losen Seite des Darms injiziert. <img alt="Abbildung 3" src="/files/ftp_upload/3225/3225fig3.jpg" /> <strong&gt; Abbildung 3. Berg der Proben. Die Proben wurden in eine 30% ige Glycerin-Lösung in PBS auf eine spezielle Folie, in der eine perforierte Kunststoffplatte (1mm dick) durch Kleber auf einem Glasträger gebunden war eingebettet. <img alt="Abbildung 4" src="/files/ftp_upload/3225/3225fig4.jpg" /> Abbildung 4. Beispiel einer Maus ligiert Peyer-Patch Loop-Test mit GFP-S. typhimurium. GP2, die als M-Zell-spezifische Marker 5 berichtet wird, wurde mit anti-Maus-Antikörper GP2 (Red) gefärbt. Die Probe wurde mit einer DeltaVision Restoration Dekonvolution Mikroskop beobachtet. GFP-exprimierenden S. Typhimurium wurde mit GP2 auf der apikalen Plasmamembran der M-Zelle und in der subapicalen zytoplasmatischen Vesikeln (Pfeilspitzen) co-lokalisiert. Top, apikale Ansicht. Unten und seitlich, seitliche Ausblicke. Maßstab: 10 pm.

<ol>
	<li>Fagarasan, S., Honjo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+IgA+synthesis:+regulation+of+front-line+body+defences.">Intestinal IgA synthesis: regulation of front-line body defences.</a> Nat. Rev. Immunol. 3, 63-72 (2003).</li><li>Hapfelmeier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+microbial+colonization+of+germ-free+mice+reveals+the+dynamics+of+IgA+immune+responses.">Reversible microbial colonization of germ-free mice reveals the dynamics of IgA immune responses.</a> Science. 328, 1705-1709 (2010).</li><li>Owen, R. L., Jones, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+specialization+within+human+Peyer's+patches:+an+ultrastructural+study+of+intestinal+lymphoid+follicles.">Epithelial cell specialization within human Peyer's patches: an ultrastructural study of intestinal lymphoid follicles.</a> Gastroenterology. 66, 189-203 (1974).</li><li>Neutra, M. R., Mantis, N. J., Kraehenbuhl, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collaboration+of+epithelial+cells+with+organized+mucosal+lymphoid+tissues.">Collaboration of epithelial cells with organized mucosal lymphoid tissues.</a> Nat. Immunol. 2, 1004-1009 (2001).</li><li>Hase, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+through+glycoprotein+2+of+FimH(+)+bacteria+by+M+cells+initiates+mucosal+immune+response.">Uptake through glycoprotein 2 of FimH(+) bacteria by M cells initiates mucosal immune response.</a> Nature. 462, 226-230 (2009).</li><li>Punyashthiti, K., Finkelstein, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteropathogenicity+of+Escherichia+coli.+I.+Evaluation+of+mouse+intestinal+loops.">Enteropathogenicity of Escherichia coli. I. Evaluation of mouse intestinal loops.</a> Infect. Immun. 4, 473-478 (1971).</li><li>Owen, R. L., Pierce, N. F., Apple, R. T., Cray, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M+cell+transport+of+Vibrio+cholerae+from+the+intestinal+lumen+into+Peyer's+patches:+a+mechanism+for+antigen+sampling+and+for+microbial+transepithelial+migration.">M cell transport of Vibrio cholerae from the intestinal lumen into Peyer's patches: a mechanism for antigen sampling and for microbial transepithelial migration.</a> J. Infect. Dis. 153, 1108-1118 (1986).</li><li>Owen, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M+cells--entryways+of+opportunity+for+enteropathogens.">M cells--entryways of opportunity for enteropathogens.</a> J. Exp. Med. 180, 7-9 (1994).</li><li>Clark, M. A., Hirst, B. H., Jepson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M-cell+surface+beta1+integrin+expression+and+invasin-mediated+targeting+of+Yersinia+pseudotuberculosis+to+mouse+Peyer's+patch+M+cells.">M-cell surface beta1 integrin expression and invasin-mediated targeting of Yersinia pseudotuberculosis to mouse Peyer's patch M cells.</a> Infect. Immun. 66, 1237-1243 (1998).</li><li>Jensen, V. B., Harty, J. T., Jones, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+the+invasive+pathogens+Salmonella+typhimurium,+Listeria+monocytogenes,+and+Shigella+flexneri+with+M+cells+and+murine+Peyer's+patches.">Interactions of the invasive pathogens Salmonella typhimurium, Listeria monocytogenes, and Shigella flexneri with M cells and murine Peyer's patches.</a> Infect. Immun. 66, 3758-3766 (1998).</li><li>Nagai, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Peyer's+patches+in+the+induction+of+Helicobacter+pylori-induced+gastritis.">Role of Peyer's patches in the induction of Helicobacter pylori-induced gastritis.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 8971-8976 (2007).</li></ol>

Keine Interessenskonflikte erklärt.

<html> Die Inkubationszeit von ligiert Peyer-Patch mit Bakteriensuspension ist in der Regel für 1 Stunde, um die bakterielle Einbau in M-Zellen zu beobachten. Im Falle von 4 Stunden Inkubation werden die Bakterien oft in der T-Zell-Zone der Peyer-Plaques nachgewiesen. Wie das verdampfte Isofluran inhalant Anästhesie halten konnte Mäusen stabil ist, ist die Inkubationszeit von ligiert Peyer-Patch mit Bakteriensuspension erweiterbar, um die Bakterien, die in T-Zell-Zone in die Peyer-Patch bewegen zu beobachten. Der wich...

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare (optional) </th></tr><tr><td> LB Agar Ampicillin- 100 </td><td> SIGMA </td><td> L5667 </td><td></td></tr><tr><td> Cy3 mono-Reactive Dye-Pack </td><td> GE Healthcare </td><td> PA23001 </td><td></td></tr><tr><td> Alexa Fluor 350 Carbonsäure, Succinimidylester </td><td> Invitrogen </td><td> A10168 </td><td></td></tr><tr><td> Inhalationsnarkose Apparat </td><td> Shinano Seisakusho </td><td> SN-487 </td><td></td></tr><tr><td> Fixation und Permeabilisierung Lösung </td><td> BD </td><td> 554722 </td><td></td></tr><tr><td> Anti-Maus Glycoprotein 2 Antikörper </td><td> MBL </td><td>D278-3 </td><td> 200-facher Verdünnung (5μg/ml) </td></tr><tr><td> Goat Anti-Rat IgG, F (ab &#39;) 2-Fragment spezifisch </td><td> Jackson ImmunoResearch </td><td> 112-505-006 </td><td> 200-facher Verdünnung (20μg/ml) </td></tr><tr><td> Alexa Fluor 633 Phalloidin </td><td> Molecular Probes </td><td> A22284 </td><td> 50-facher Verdünnung </td></tr><tr><td> Konfokalen Laser Scanning-Mikroskop </td><td> Leica Microsystems </td><td> DMIRE2 </td><td></td></tr><tr><td> DeltaVision Restaurierung Dekonvolution Mikroskop </td><td> Applied Precision </td><td> DeltaVision Core- </td><td></td></tr></tbody></table>

application of a mouse ligated peyer&#x2019;s patch intestinal loop assay to evaluate bacterial uptake by m cells

Das Innere unseres Darms ist mit enormen Anzahl von symbiotischer Bakterien besiedelt. Die Schleimhautoberfläche des Magen-Darm-Trakt ist ständig mit ihnen und gelegentlich, um Krankheitserreger ausgesetzt. Der Darm-assoziierten lymphatischen Gewebe (GALT) spielen eine Schlüsselrolle für die Induktion der mukosalen Immunantwort auf diese Mikroben 1, 2. Zur Initiierung der mukosalen Immunantwort, muss die Schleimhautzellen Antigene aus dem Darmlumen in das Epithel in organisierte lymphoide Follikel, wie Peyer-Plaques transportiert werden. Dieses Antigen Transzytose wird von spezialisierten epithelialen Zellen M 3, 4 vermittelt. M-Zellen sind atypische Epithelzellen, die sich aktiv phagozytieren Makromoleküle und Mikroben. Im Gegensatz zu dendritischen Zellen (DCs) und Makrophagen, die Ziel-Antigene zu Lysosomen zum Abbau, M-Zellen hauptsächlich transcytose die verinnerlichten Antigene. Diese kräftige makromolekularen Transzytose durch M-Zellen liefert Antigen auf die zugrunde liegende organisierte lymphoide Follikel eind ist vermutlich wesentlich für die Einleitung Antigen-spezifische mukosale Immunantworten. Allerdings sind die molekularen Mechanismen der Förderung dieses Antigen-Aufnahme durch M-Zellen weitgehend unbekannt. Wir haben bereits berichtet, dass Glykoprotein 2 (GP2), insbesondere auf der apikalen Plasmamembran der M-Zellen unter Enterozyten exprimiert, als transcytotic Rezeptor für eine Teilmenge von Kommensalen und pathogene Enterobakterien, darunter Escherichia coli und Salmonella enterica Serovar Typhimurium (S. typhimurium dient ), durch die Anerkennung FimH, eine Komponente des Typ-I-Pili auf der bakteriellen äußeren Membran 5. Hier stellen wir eine Methode für die Anwendung einer Maus Peyer-Patch Darmschlinge Test auf bakterielle Aufnahme durch M-Zellen zu bewerten. Diese Methode ist eine verbesserte Version der Maus Darmschlinge Assay zuvor beschriebenen 6, 7. Die verbesserte Punkte sind wie folgt: 1. Isofluran wurde als Narkotikum verwendet. 2. Etwa 1 cm ligiert Darmschlinge einschließlichIng. Peyer-Patch wurde eingerichtet. 3. Bakterien durch M-Zellen aufgenommen wurden Fluoreszenz durch Fluoreszenzmarkierung Reagenz oder durch Überexpression fluoreszierenden Proteins, wie grün fluoreszierende Protein (GFP) markiert. 4. M-Zellen in der Follikel-assoziierte Epithel Peyer-Patch wurden von insgesamt-mount immunostainig mit anti Gp2 Antikörper nachgewiesen. 5. Fluoreszierende Bakterien Transzytose von M-Zellen wurden durch konfokale mikroskopische Analyse beobachtet. Die Maus Peyer-Patch Darmschlinge Test liefern könnte die Antwort, welche Art von Kommensalen oder pathogenen Bakterien durch M-Zellen transcytosed und kann uns dazu bringen, den molekularen Mechanismus, wie mukosalen Immunsystems durch M-Zellen zu stimulieren verstehen.

Watch this Scientific Journal Video about Application of a Mouse Ligated Peyerâ€™s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells at JoVE.com

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M-Zellen in einem spezialisierten Follikel-assoziierte Epithel Peyer-Plaques eine wichtige Rolle spielen für die Schleimhaut Immunüberwachung im Darm-assoziierten lymphatischen Gewebe. Hier beschreiben wir die Auswertungsverfahren für die bakterielle Transzytose von M-Zellen<em&gt; In vivo</em&gt;. Diese Methode stellt eine Methode zur M-Zell-Funktion im Immunsystem zu verstehen.

Die Anwendung eines Maus Ligierte Peyer-Patch-Darmschlinge Assay zu bakterielle Aufnahme von M-Zellen auswerten

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

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Research

JoVE Journal

Immunology and Infection

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

19.4K Aufrufe.  RIKEN Research Center for Allergy and Immunology. M-Zellen in einem spezialisierten Follikel-assoziierte Epithel Peyer-Plaques eine wichtige Rolle spielen für die Schleimhaut Immunüberwachung im Darm-assoziierten lymphatischen Gewebe. Hier beschreiben wir die Auswertungsverfahren für die bakterielle Transzytose von M-Zellen In vivo. Diese Methode stellt eine Methode zur M-Zell-Funktion im Immunsystem zu verstehen.

Serie: Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Mit Luciferase to Image Bakterielle Infektionen bei Mäusen

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Forschung

Immunologie und Infektion

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

Invasion von menschlichen Zellen durch einen bakteriellen Erreger

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

Ein Visual Assay zur T6SS-vermittelte bakterielle Wettbewerb überwachen

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g., diets, antigens, drugs) and in disease (e.g., inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

Die Mesenteriallymphknoten Duct Cannulated Rattenmodell: Anwendung auf die Bewertung der Darm Lymphatische Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Application of Long-term cultured Interferon-&amp;#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

Anwendung von Langzeit kultiviert Interferon-γ Enzyme-linked Immunospot Assay zur Beurteilung Effektor und Gedächtnis-T-Zell-Antworten in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Die Verwendung eines Monozyten einschichtiges Assay Fcy rezeptorvermittelte Phagozytose zur Bewertung

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

Immunfluoreszenzanalyse der Stressgranulat-Bildung nach bakterieller Herausforderung von Säugetierzellen

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

Eine neuartige In-vitro- Wunde heilende Assay Zellwanderung bewerten

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

Cell Membrane Repair Assay Using a Two-photon Laser Microscope

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can result in disease. Understanding the underlying molecular mechanisms surrounding cell membrane repair is, therefore, an important objective to the development of novel therapeutic strategies for diseases associated with dysfunctional cell membrane dynamics. Many in vitro and in vivo studies aimed at understanding cell membrane resealing in various disease contexts utilize two-photon laser ablation as a standard for determining functional outcomes following experimental treatments. In this assay, cell membranes are subjected to wounding with a two-photon laser, which causes the cell membrane to rupture and fluorescent dye to infiltrate the cell. The intensity of fluorescence within the cell can then be monitored to quantify the cell's ability to reseal itself. There are several alternative methods for assessing cell membrane response to injury, as well as great variation in the two-photon laser wounding approach itself, therefore, a single, unified model of cell wounding would beneficially serve to decrease the variation between these methodologies. In this article, we outline a simple two-photon laser wounding protocol for assessing cell membrane repair in vitro in both healthy and dysferlinopathy patient fibroblast cells transfected with or without a full-length dysferlin plasmid.

Cell membrane wounding via two-photon laser is a widely used method for assessing membrane resealing ability and can be applied to multiple cell types. Here, we describe a protocol for in vitro live-imaging of membrane resealing in dysferlinopathy patient cells following two-photon laser ablation.

Zellmembran Reparatur Assays mit einem zwei-Photonen-Laser-Mikroskop

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can ...

Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water environment. Developing effective methods for oral delivery of immunostimulants or vaccines, which activate the immune system against infectious diseases, is highly desirable. In devising prophylactic tools, good experimental models are needed to test their performance. Here, we show a method for oral intubation of adult zebrafish and a set of procedures to dissect and prepare the intestine for cytometry, confocal microscopy and quantitative polymerase chain reaction (qPCR) analysis. With this protocol, we can precisely administer volumes up to 50 &#181;L to fish weighing approximately 1 g simply and quickly, without harming the animals. This method allows us to explore the direct in vivo uptake of fluorescently labelled compounds by the intestinal mucosa and the immunomodulatory capacity of such biologics at the local site after intubation. By combining downstream methods such as flow cytometry, histology, qPCR and confocal microscopy of the intestinal tissue, we can understand how immunostimulants or vaccines are able to cross the intestinal mucosal barriers, pass through the lamina propria, and reach the muscle, exerting an effect on the intestinal mucosal immune system. The model could be used to test candidate oral prophylactics and delivery systems or the local effect of any orally administered bioactive compound.

The protocol describes intubating adult zebrafish with a biologic; then dissecting and preparing the intestine for cytometry, confocal microscopy and qPCR. This method allows administration of bioactive compounds to monitor intestinal uptake and the local immune stimulus evoked. It&#160;is relevant for testing the intestinal dynamics of oral prophylactics.