Name: isolation, characterization and functional examination of the gingival immune cell network
Uploaded: 2016-02-16T15:00:00
Description: Watch this Scientific Journal Video about Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network at JoVE.com

Authors were funded in part by the intramural program of NIDCR (N.M.M) and supported by a Wellcome Trust Stepping Stones Fellowship (097820/Z/11/B to J.E.K) and by a Manchester Collaborative Centre for Inflammation Research grant (to J.E.K). The authors thank Teresa Wild for critically reviewing the manuscript.

Alle eksperimentelle prosedyrer som er beskrevet i denne protokollen følges nødvendige retningslinjer og ble godkjent av Institutional Animal Care og bruk komité, NIDCR / NIH. 1. Forbered på forhånd <ol><li> Forbered Komplett media: RPMI supplert med 2 mM L-glutamin, 100 enheter / ml penicillin, 100 ug / ml streptomycin og 10% FBS. </li><li> Forbered DNase media: 50 ml RPMI media supplert med 7,5 mikrogram DNase (Gjør frisk, holde på is til alle tider). </li><li> Forbered Collage-DNase media: 5 ml DNase...

<ol>
	<li>Aas, J. A., Paster, B. J., Stokes, L. N., Olsen, I., Dewhirst, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+The+Normal+Bacterial+Flora+Of+The+Oral+Cavity.">Defining The Normal Bacterial Flora Of The Oral Cavity.</a> J Clin Microbiol. 43, 5721-5732 (2005).</li><li>Belkaid, Y., Naik, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalized+And+Systemic+Control+Of+Tissue+Immunity+By+Commensals.">Compartmentalized And Systemic Control Of Tissue Immunity By Commensals.</a> Nat Immunol. 14, 646-653 (2013).</li><li>Darveau, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periodontitis:+A+Polymicrobial+Disruption+Of+Host+Homeostasis.">Periodontitis: A Polymicrobial Disruption Of Host Homeostasis.</a> Nat Rev Microbiol. 8, 481-490 (2010).</li><li>Hajishengallis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomicrobial+Pathogenesis+Of+Periodontitis:+Keystones,+Pathobionts,+And+Host+Response.">Immunomicrobial Pathogenesis Of Periodontitis: Keystones, Pathobionts, And Host Response.</a> Trends Immunol. 35, 3-11 (2014).</li><li>Eke, P. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+Of+Periodontitis+In+Adults+In+The+United+States:+2009+And+2010.">Prevalence Of Periodontitis In Adults In The United States: 2009 And 2010.</a> J Dent Res. 91, 914-920 (2012).</li><li>Moutsopoulos, N. M., Lionakis, M. S., Hajishengallis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inborn+Errors+In+Immunity:+Unique+Natural+Models+To+Dissect+Oral+Immunity.">Inborn Errors In Immunity: Unique Natural Models To Dissect Oral Immunity.</a> J Dent Res. , (2015).</li><li>Hajishengallis, G., Lamont, R. J., Graves, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Enduring+Importance+Of+Animal+Modelsin+Understanding+Periodontal+Disease.">The Enduring Importance Of Animal Modelsin Understanding Periodontal Disease.</a> Virulence. 6, 229-235 (2015).</li><li>Sharrow, S. O., Coligan, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+Of+Flow+Cytometry+Data.">Analysis Of Flow Cytometry Data.</a> Current Protocols In Immunology. , (2001).</li><li>Arizon, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Langerhans+Cells+Down-Regulate+Inflammation-Driven+Alveolar+Bone+Loss.">Langerhans Cells Down-Regulate Inflammation-Driven Alveolar Bone Loss.</a> Proc Natl Acad Sci U S A. 109, 7043-7048 (2012).</li><li>Moutsopoulos, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+Neutrophil+Recruitment+In+Leukocyte+Adhesion+Deficiency+Type+I+Disease+Causes+Local+IL-17-Driven+Inflammatory+Bone+Loss.">Defective Neutrophil Recruitment In Leukocyte Adhesion Deficiency Type I Disease Causes Local IL-17-Driven Inflammatory Bone Loss.</a> Sci Transl Med. 6, 229-240 (2014).</li><li>Gaffen, S. L., Jain, R., Garg, A. V., Cua, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+IL-23-IL-17+Immune+Axis:+From+Mechanisms+To+Therapeutic+Testing.">The IL-23-IL-17 Immune Axis: From Mechanisms To Therapeutic Testing.</a> Nat Rev Immunol. 14, 585-600 (2014).</li></ol>

Den nåværende teknikk med hell gir et stort antall av immunceller (fra en enkelt mus) passende, ikke bare for fenotypisk karakterisering, men også for funksjonelle studier ex vivo. En annen gruppe hadde tidligere publisert en protokoll for å isolere og karakterisere murine gingivale immunceller og innføres verdien av å bruke flerfarget flowcytometri i studiet av periodontitt i dyremodeller 9. Etter betydelig feilsøking nøkkelen endring i denne protokollen var å inkludere disseksjon av hele b...

Gingivalt vev omgir den humane og murine tannsett og er stadig utsatt for det komplekse biofilm av tannen 1. Den immuncellenettverket politi gingival barrieren er viktig å opprettholde vev integritet, noe som sikrer homeostase med de lokale kommensale mikroorganismer og, på samme tid, noe som gir effektiv immunitet mot sykdomsfremkallende utfordring 2. For å oppnå homeostase, immunsystemet nøye skreddersydd til gingival miljøet skape et høyt spesialisert immun celle nettverk, men liten detalj er kjent av tannkjøttimmuncellepopulasjoner og deres rolle i å opprettholde vev immunitet to. Når immun homeostase blir forstyrret på gingiva, enten gjennom økt vert mottakelighet og / eller tilstedeværelse av dysbiotic mikrobielle samfunn, en betennelsestilstand, oppstår periodontitt 3 4. Periodontitt er en vanlig inflammatorisk sykdom, som fører til tap av tann supporting strukturer. I sin alvorlige former er det sett hos ca. 10% av befolkningen fem. Dissekere de viktigste faktorene som er involvert i periodontitt mottakelighet og progresjon har vist seg vanskelig seks. Imidlertid har dyremodeller vært svært nyttig for å forstå mekanismene for periodontitt initiering og progresjon 7. Modeller kan benyttes for å definere de viktigste cellepopulasjoner og molekylære meklere som er avgjørende for å opprettholde immun homeostase og drive utvikling av periodontitt. En slik innsikt vil forandre vår forståelse av gingiva spesifikk kontroll av immun homeostase og videreutvikle vår nåværende forståelse av sykdom patogenesen.

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isolation, characterization and functional examination of the gingival immune cell network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

For å illustrere anvendelsen av protokollen, viser vi representative resultater undersøke immuncellenettverket i gingiva hos mus med og uten periodontitt (WT vs. LFA - / -, Figur 2A - C). Representative FACS tomter vise live-CD45 + hematopoetiske celler i gingiva (figur 2A, 2C). Isolering og behandling av immunceller med denne protokollen gir tilstrekkelig antall celler for å utføre ex vivo<...

Watch this Scientific Journal Video about Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network at JoVE.com

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Vi har etablert en teknikk for isolering, fenotypiske karakterisering og funksjonell analyse av immunceller fra mus gingiva.

Isolasjon, karakterisering og Funksjonell Undersøkelse av Gingival immun Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

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