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.

Tutte le procedure sperimentali descritte in questo protocollo seguite le linee guida necessarie e sono stati approvati dalla cura degli animali e del Comitato Istituzionale Usa, NIDCR / NIH. 1. preparare in anticipo <ol><li> Preparare supporti Completa: RPMI integrato con 2 mM L-glutammina, 100 unità / ml di penicillina, 100 ug / ml di streptomicina e il 10% FBS. </li><li> Preparare supporti DNase: 50 ml di media RPMI con 7,5 mcg DNasi (Fai fresca, tenere in ghiaccio in ogni momento). </li><li> Preparare sup...

<ol>
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L&#39;attuale tecnica produce con successo un gran numero di cellule immunitarie (da un singolo mouse) adatto, non solo per la caratterizzazione fenotipica, ma anche per studi funzionali ex vivo. Un altro gruppo aveva già pubblicato un protocollo per isolare e caratterizzare le cellule immunitarie murino gengivali e ha introdotto il valore di usare multicolore citometria a flusso nello studio di parodontite in modelli animali 9. A seguito di una notevole risoluzione dei problemi della modifica chiav...

Tessuti gengivali circondano la dentatura murino e umano e sono costantemente esposti al complesso biofilm del dente 1. La rete di cellule immunitarie sorvegliare la barriera gengivale è fondamentale per mantenere l&#39;integrità del tessuto, garantendo omeostasi con i microbi commensali locali e, al tempo stesso, assicura immunità efficace contro sfida patogenicità 2. Per raggiungere l&#39;omeostasi, il sistema immunitario è attentamente su misura per l&#39;ambiente gengivale creazione di una rete di cellule immunitarie altamente specializzato, ma piccolo dettaglio si sa delle popolazioni di cellule immunitarie gengivali e il loro ruolo nel mantenimento del tessuto immunità 2. Quando l&#39;omeostasi immunitaria è interrotta alla gengiva, sia attraverso una maggiore suscettibilità dell&#39;ospite e / o la presenza di comunità microbiche disbiosico, una condizione infiammatoria, la parodontite si pone 3 4. La parodontite è una malattia infiammatoria comune, con conseguente perdita del dente sstrutture upporting. Nelle sue forme più gravi si è visto in circa il 10% della popolazione generale 5. Analisi dei fattori chiave coinvolti nella predisposizione e nella progressione della parodontite si è dimostrato difficile 6. Tuttavia, i modelli animali sono stati estremamente utili per la comprensione dei meccanismi di iniziazione parodontite e la progressione 7. I modelli possono essere utilizzati per definire le popolazioni di cellule chiave e mediatori molecolari che sono vitali per il mantenimento dell&#39;omeostasi immunitario e lo sviluppo di auto di periodontite. Tale intuizione trasformerà la nostra comprensione di controllo specifico-gengiva di omeostasi immunitaria e ulteriormente la nostra attuale comprensione della patogenesi della malattia.

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			<td height="21" style="height:21px;width:304px;">70 &#956;M Cell Strainers</td>
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			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD11c FITC</td>
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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.

Per illustrare l&#39;applicazione del protocollo, si mostra risultati rappresentativi esaminando la rete delle cellule immunitarie nella gengiva di topi con e senza parodontite (WT vs LFA - / -, Figura 2a - C). FACS Rappresentante grafici mostrano le cellule CD45 + ematopoietiche dal vivo nella gengiva (Figura 2A, 2C). L&#39;isolamento e la trasformazione delle cellule immunitarie con questo protoco...

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

Abbiamo stabilito una tecnica per l&#39;isolamento, la caratterizzazione fenotipica e analisi funzionale di cellule immuni dalla gengiva murino.

Isolamento, caratterizzazione e esame funzionale della cella di rete gengivale immunitario

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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