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 ønsker å takke alle EIB medlemmer for å utvikle denne teknikken. Denne forskningen ble støttet delvis av Grant-i-Aid for vitenskapelig forskning på prioriterte områder &quot;Membrane Traffic&quot; (HO), og &quot;Matrix of Infectious Phenomena&quot; (KH), Unge Forskere (SF og KH) og Scientific Research (HO ), og forskning på innovative områder &quot;Intracellulær Logistics&quot; (HO) fra Ministry of Education, Culture, Sports, Science and Technology of Japan.

1. Utarbeidelse av bakterieceller <ol><li> Streak glyserol lager selvlysende bakterier (som GFP uttrykke S. Typhimurium) på LB en agar plate som inneholder 100 mikrogram / ​​ml ampicillin. </li><li> Kultur én koloni fra LB agar natten i 2 ml av nye LB medium. </li><li> Tilsett 0,5 ml av bakteriell kultur til 4,5 ml av nye LB medium og inkuber til optisk tetthet på 1,0 på 600 nm er nådd. </li><li> Høste bakterieceller ved sentrifugering (3000 xg, 5 min, 4 ° C). </li><li> Kast supernatanten og vask t...

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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> Inkubasjonstiden for ligated Peyer sin lapp med bakteriell suspensjon er vanligvis for 1 time å observere den bakterielle innlemmelse i M celler. Ved 4 timer inkubering, er bakteriene ofte påvist i T-celle sonen Peyer sin patcher. Som fordampede isofluran inhalant anestesi kunne holde mus stabil, er det inkubasjonstid på ligated Peyer sin lapp med bakteriell suspensjon utvides til å observere bakterier som flytter inn i T-celle-sonen i Peyer sin patch. Det viktige poenget med dette eksperimentet er å ta forsi...

<table border="1"><tbody><tr><th> Navn på reagens </th><th> Firma </th><th> Katalognummer </th><th> Kommentarer (valgfritt) </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 karboksylsyre, succinimidyl ester </td><td> Invitrogen </td><td> A10168 </td><td></td></tr><tr><td> Innånding anestesi apparater </td><td> Shinano Seisakusho </td><td> SN-487 </td><td></td></tr><tr><td> Fiksering og Permeabilization Oppløsning </td><td> BD </td><td> 554722 </td><td></td></tr><tr><td> Anti mus Glykoprotein 2 antistoff </td><td> MBL </td><td>D278-3 </td><td> 200-fold fortynning (5μg/ml) </td></tr><tr><td> Goat Anti-Rat IgG, F (ab &#39;) 2 fragment spesifikke </td><td> Jackson ImmunoResearch </td><td> 112-505-006 </td><td> 200-fold fortynning (20μg/ml) </td></tr><tr><td> Alexa Fluor 633 Phalloidin </td><td> Molekylære prober </td><td> A22284 </td><td> 50-fold fortynning </td></tr><tr><td> Confocal laser scanning mikroskop </td><td> Leica Microsystems </td><td> DMIRE2 </td><td></td></tr><tr><td> DeltaVision Restaurering deconvolution mikroskop </td><td> Applied Precision </td><td> DeltaVision Kjerne </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

Innsiden av tarmen vår er bebodd med enorme antall commensal bakterier. Mucosal overflaten av gastrointestinaltraktus er kontinuerlig utsatt for dem og noen ganger til patogener. Tarmen-assosiert lymfoid vev (Galt) spiller en nøkkelrolle for induksjon av mucosal immunresponsen på disse mikrobene 1, 2. For å starte mucosal immunrespons, må mucosal antigener skal transporteres fra tarmen lumen over epiteliale barriere i organiserte lymfoide follikler som Peyer er patcher. Dette antigen transcytosis er mediert av spesialiserte epitelceller M celler 3, 4. M celler er atypiske epiteliale celler som aktivt phagocytose makromolekyler og mikrober. I motsetning til dendrittiske celler (DCS) og makrofager som mål antigener til lysosomer for nedbrytning, M celler hovedsakelig transcytose den internaliserte antigener. Dette livskraftige macromolecular transcytosis gjennom M celler leverer antigen til den underliggende organiserte lymfoide follikler end antas å være avgjørende for å initiere antigen-spesifikke slimhinnene immunrespons. Men de molekylære mekanismene som fremmer dette antigen opptak av M celler er i stor grad ukjent. Vi har tidligere rapportert at glykoprotein 2 (GP2), spesifikt uttrykt på den apikale plasma membran av M celler blant enterocytter, fungerer som et transcytotic reseptor for en undergruppe av commensal og patogene enterobacteria, inkludert Escherichia coli og Salmonella enterica serovar Typhimurium (S. Typhimurium ), ved å anerkjenne FimH, en komponent av type I pili på bakteriens ytre membran 5. Her presenterer vi en metode for anvendelse av en mus Peyer er patch intestinal sløyfe analysen å vurdere bakteriell opptak av M celler. Denne metoden er en forbedret versjon av musa intestinal sløyfe analysen tidligere beskrevet 6, 7. Den forbedrede punktene er som følger: 1. Isofluran ble brukt som anestesimiddel. 2. Ca 1 cm ligated intestinal sløyfe inkluing Peyer sin patch ble satt opp. 3. Bakterier tatt opp av M celler ble fluorescensmerkede av fluorescens merking reagens eller ved overekspresjon fluorescerende protein som grønn fluorescerende protein (GFP). Fire. M cellene i hårsekken-assosiert epitel dekker Peyer sin lapp ble oppdaget av hel-mount immunostainig med anti GP2 antistoff. 5. Fluorescent bakteriell transcytosis av M celler ble observert av confocal mikroskopisk analyse. Musen Peyer er patch intestinal sløyfe analysen kunne levere svaret hva slags commensal eller sykdomsfremkallende bakterier transcytosed av M celler, og kan lede oss til å forstå den molekylære mekanismen for hvordan å stimulere slimhinnene immunforsvaret gjennom 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 et spesialisert follicle-assosiert epitel dekker Peyer er patcher spiller en viktig rolle for mucosal immunosurveillance i gut-assosiert lymfoid vev. Her har vi beskrev evalueringsmetode for bakteriell transcytosis av M celler<em&gt; In vivo</em&gt;. Denne metoden gir en metode for å forstå M-celle funksjon i immunsystemet.

Anvendelse av en Mouse Ligated Peyer er Patch Intestinal Loop Assay å evaluere Bakteriell Opptak av 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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