Name: application of a mouse ligated peyer&#x2019;s patch intestinal loop assay to evaluate bacterial uptake by m cells
Uploaded: 2011-12-17T14:00:00
Description: Watch this Scientific Journal Video about Application of a Mouse Ligated PeyerÃ¢â‚¬â„¢s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells at JoVE.com

Forfatterne vil gerne takke alle EIB-medlemmer til at udvikle denne teknik. Denne forskning blev delvist understøttet af Grant-in-Støtte til videnskabelig forskning på prioriterede områder &quot;Membran Traffic&quot; (HO), og &quot;Matrix af infektiøs Phenomena&quot; (KH), Unge Forskere (SF og KH), og videnskabelig forskning (HO ), og videnskabelig forskning i innovative områder &quot;Intracellulær Logistics&quot; (HO), fra Ministeriet for Undervisning, Kultur, Sport, Videnskab og Teknologi i Japan.

1. Udarbejdelse af bakterieceller <ol><li> Streak glycerol lager fluorescerende bakterier (som f.eks GFP udtrykke S. Typhimurium) på LB en agarplade indeholdende 100 mg / ml af ampicillin. </li><li> Kultur en enkelt koloni fra LB agar natten i 2 ml nye LB medium. </li><li> Tilsæt 0,5 ml bakteriekultur til 4,5 ml af nye LB medium og inkuber indtil optisk tæthed på 1,0 ved 600 nm er nået. </li><li> Harvest bakterieceller ved centrifugering (3.000 xg, 5 min, 4 ° C). </li><li> Supernatanten fjernes, og vask...

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	<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>

<html> Inkubationstiden for ligated Peyer patch med bakterieopløsning er normalt for 1 time at observere den bakterielle inkorporering i M-celler. I tilfælde af 4 timer inkubation, er bakterierne ofte påvises i T-celle zone Peyerske plaques. Da fordampet isofluran inhalationskapsler narkose kunne holde mus stabil, inkubationstid på ligated Peyer patch med bakteriel suspension kan udvides til at overholde de bakterier, der flytter ind i T-celle zone i Peyer patch. Det vigtige punkt i dette eksperiment er at passe på ...

<table border="1"><tbody><tr><th> Navn på reagenset </th><th> Firma </th><th> Katalog nummer </th><th> Kommentarer (valgfri) </th></tr><tr><td> LB Agar Ampicillin- 100 </td><td> SIGMA </td><td> L5667 </td><td></td></tr><tr><td> Cy3 mono-reaktivt Dye Pack </td><td> GE Healthcare </td><td> PA23001 </td><td></td></tr><tr><td> Alexa Fluor 350 carboxylsyre, succinimidyl ester </td><td> Invitrogen </td><td> A10168 </td><td></td></tr><tr><td> Indånding anæstesi apparater </td><td> Shinano Seisakusho </td><td> SN-487 </td><td></td></tr><tr><td> Fiksering og Permeabilization Løsning </td><td> BD </td><td> 554722 </td><td></td></tr><tr><td> Anti musen Glykoprotein 2 antistof </td><td> MBL </td><td>D278-3 </td><td> 200-fold fortynding (5μg/ml) </td></tr><tr><td> Goat Anti-Rat IgG, F (ab &#39;) 2 fragment specifikke </td><td> Jackson ImmunoResearch </td><td> 112-505-006 </td><td> 200-fold fortynding (20μg/ml) </td></tr><tr><td> Alexa Fluor 633 Phalloidin </td><td> Molecular Probes </td><td> A22284 </td><td> 50-fold fortynding </td></tr><tr><td> Konfokal laser scanning mikroskop </td><td> Leica Microsystems </td><td> DMIRE2 </td><td></td></tr><tr><td> DeltaVision Restaurering deconvolution mikroskop </td><td> Anvendt 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

Indersiden af ​​vores tarm er beboet med enorme antal kommensale bakterier. Slimhinden i mave-tarmkanalen er kontinuerligt udsættes for dem og lejlighedsvis for patogener. Den gut-associerede lymfoide væv (Galt) spiller en central rolle for induktion af de mucosal immun reaktion på disse mikrober 1, 2. At indlede slimhinden immunrespons, skal slimhinde antigener blive transporteret fra tarmen lumen på tværs af epithelial barrieren til organiseret lymfoide follikler som Peyerske plaques. Dette antigen transcytosis er medieret af specialiserede epitelceller M celler 3, 4. M celler er atypiske epitelceller, der aktivt phagocytose makromolekyler og mikrober. I modsætning til dendritiske celler (DCS) og makrofager, som er målrettet antigener til lysosomer for nedbrydning, hovedsageligt M celler transcytose den internaliserede antigener. Denne energiske makromolekylære transcytosis gennem M celler leverer antigen til den underliggende organiseret lymfoide follikler end menes at være af afgørende betydning for iværksættelse af antigen-specifikke mucosale immunrespons. Men de molekylære mekanismer, der fremmer dette antigen optagelse af M celler er stort set ukendte. Vi har tidligere rapporteret, at glykoprotein 2 (GP2), især udtrykt på den apikale plasma membran af M celler blandt enterocyterne, tjener som et transcytotic receptor for en delmængde af kommensale og patogene enterobakterier, herunder Escherichia coli og Salmonella enterica serovar Typhimurium (S. Typhimurium ), ved at anerkende FimH, en komponent af type I Pili på den bakterielle ydre membran 5. Her præsenterer vi en metode for anvendelse af en mus Peyer patch intestinal loop-analysen for at vurdere bakterielle optagelse af M celler. Denne metode er en forbedret udgave af musens tarm loop assay tidligere beskrevet 6, 7. De forbedrede punkter er som følger: 1. Isofluran blev brugt som et bedøvelsesmiddel agent. 2. Ca. 1 cm ligated tarm-loop herunderING Peyer patch blev oprettet. 3. Bakterier taget op af M-celler var fluorescens-mærkede ved fluorescens mærkning reagens eller ved overekspression fluorescerende protein som grønt fluorescerende protein (GFP). 4. M celler i hårsækken-associerede epitel dækker Peyer patch blev opdaget af hel-mount immunostainig med anti GP2 antistof. 5. Fluorescerende bakteriel transcytosis af M celler blev observeret ved konfokal mikroskopisk analyse. Musen Peyer patch tarm-loop-analysen kunne levere svaret, hvilken slags kommensale eller sygdomsfremkaldende bakterier transcytosed af M celler, og kan føre os til at forstå de molekylære mekanisme, hvordan man kan stimulere mucosal immunsystem gennem M celler.

Watch this Scientific Journal Video about Application of a Mouse Ligated PeyerÃ¢â‚¬â„¢s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells at JoVE.com

Application of a Mouse Ligated PeyerÃƒÂ¢Ã¢â€šÂ¬Ã¢â€žÂ¢s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M celler i en specialiseret follikel-associerede epitel dækker Peyerske plaques spiller en vigtig rolle for mucosal immunosurveillance i tarmen-associerede lymfoide væv. Her har vi beskrev evalueringsmetode for bakteriel transcytosis af M celler<em&gt; In vivo</em&gt;. Denne metode giver en metode til at forstå M-celle funktion i immunsystemet.

Anvendelse af en mus Ligated Peyer er Patch Intestinal Loop Assay at evaluere Bakteriel Optagelse ved M celler

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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Immunology and Infection

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

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