Method Article

Isolatie, karakterisering en functionele onderzoek van het tandvlees Immune Cell Network

DOI:

10.3791/53736

⸱

February 16th, 2016

 ,  ,  , 
1Oral Immunity and Inflammation Unit, NIDCR, NIH, 2Manchester Immunology Group, Faculty of Life Sciences, University of Manchester, 3Manchester Collaborative Centre for Inflammation Research, University of Manchester

In This Article

Summary

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We hebben een techniek ontwikkeld voor de isolatie, karakterisatie fenotypische en functionele analyse van immuuncellen van muizen gingiva vastgesteld.

Abstract

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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.

Introduction

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Gingivale weefsel rondom de menselijke en murine gebit en worden voortdurend blootgesteld aan het complex biofilm van de tand 1. De immuuncel netwerk toezicht op de gingivale barrière is essentieel voor het handhaven weefselintegriteit, waardoor homeostase met de lokale commensale microben en, tegelijkertijd, doeltreffende immuniteit tegen pathogene challenge 2. Om homeostase, wordt het immuunsysteem nauwkeurig afgestemd op de gingivale milieu waardoor een sterk gespecialiseerde immuuncel netwerk, maar weinig detail bekend van de gingivale immuuncelpopulaties en hun rol bij het ​​handhaven weefsel immuniteit 2.

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Protocol

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Alle in dit protocol beschreven experimentele gevolgde procedures vereiste richtlijnen en werden goedgekeurd door de Institutional Animal Care en gebruik Comite, NIDCR / NIH.

1. Bereid in Advance

  1. Bereid compleet medium: RPMI aangevuld met 2 mM L-glutamine, 100 eenheden / ml penicilline, 100 ug / ml streptomycine en 10% FBS.
  2. Bereid DNase media: 50 ml RPMI medium aangevuld met 7,5 ug DNase (Voeg verse, houdt op ijs te allen tijde).
  3. Bereid Collagenase-DNase media: 5 ml DNase media plus 3,2 mg / ml van Collagenase Type IV (Voeg verse, houdt op ijs te allen tijde).
  4. Pre-cool FACS buffer: 0,5% FBS verdund in PBS.
  5. Pr....

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Results

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De toepassing van het protocol illustreren, tonen we representatieve resultaten onderzoeken de immuuncel netwerk in de gingiva van muizen met en zonder parodontitis (WT versus LFA - / - figuur 2A - C). Representatieve FACS plots tonen levende CD45 + hematopoietische cellen in de gingiva (Figuur 2A, 2C). Isolatie en verwerking van immuuncellen met dit protocol levert voldoende cellen ex vivo.......

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Discussion

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De huidige techniek succes een groot aantal immuuncellen (van één enkele muis) geschikt oplevert, niet alleen voor de fenotypische karakterisatie, maar ook voor functionele studies ex vivo. Een andere groep eerder gepubliceerd protocol voor het isoleren en karakteriseren van muizen gingivale immuuncellen en introduceerde de waarde van het gebruik multicolor flowcytometrie in de studie van parodontitis in diermodellen 9. Na aanzienlijke problemen de belangrijkste wijziging in dit protocol werd dissect.......

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Disclosures

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Authors have nothing to disclose

Acknowledgements

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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.

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Fine ScissorsFine science tools14058-11
Scalpel Handle #3Fine science tools10003-12
Scalpel Blades #10Fine science tools10010-00Sterile
Splinter ForcepsIntegra Miltex6-304
Needles with regular bevel BD Medical30510927 G, 12.7 mm length
Monoject syringesCovidien8881513934Luer-lock tip, 3 ml
PBS, pH 7.4Life Technologies10010-049Without Calcium and Magnesium
RPMI 1640Lonza12-167FWithout L-glutamine
DNase I from bovine pancreasSigma-AldrichDN25-1G
Collagenase type IVGibco (by Life technologies)17104-019
Fetal Bovine SerumGemini Bio-products100-106
Gentamicin 50 mg/mlQuality biological120-098-661EA
Pen StrepGibco (by Life technologies)15140-122
L-GlutamineGibco (by Life technologies)25030-081
0.5 M EDTA pH 8.0Quality biological351-027-721EA
50 ml tubesCorning352070Polypropylene, sterile
70 μM Cell StrainersCorning352350
Petri dishesCorning351029Sterile
5 ml FACS tubesCorning352052Sterile
BD GolgiPlugBD Biosciences555029Contains brefeldin A solution
Phorbol 12-Myristate 13-Acetate (PMA)Sigma-AldrichP8139
Ionomycin Calcium SaltSigma-Aldrich13909
Saponin from quillaja barkSigma-AldrichS4521
LIVE/DEAD Fixable Aqua Dead Cell Stain KitLife TechnologiesL34957
Anti-Mouse CD45 Alexa Fluor 700eBioscience56-0451-82
Anti-Mouse CD4 eFluor 450eBioscience48-0042-82
Anti-Mouse TCR beta APC eFluor 780eBioscience47-5961-82
Anti-Mouse gamma delta TCR FITCeBioscience11-5711-82
Anti-Mouse IL-17A APCeBioscience12-7311-82
Anti-Mouse IFN-γ PEeBioscience11-5931-82
Anti-Mouse NK1.1 PE-Cy7eBioscience25-5941-82
Anti-Mouse CD90.2 APC eFluor 780eBioscience47-0902-82
Anti-Mouse CD3e FITCeBioscience11-0031-82
Anti-Mouse CD19 FITCeBioscience11-0193-82
Anti-Mouse CD11b FITCeBioscience11-0112-82
Anti-Mouse CD11c FITCeBioscience11-0114-82
Anti-Mouse TCR beta FITCeBioscience11-5961-82
Anti-Mouse Ly-6G FITCeBioscience11-5931-82
Anti-Mouse Ly-6C FITCBD Pharmingen553104

References

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  1. Aas, J. A., Paster, B. J., Stokes, L. N., Olsen, I., Dewhirst, F. E. Defining The Normal Bacterial Flora Of The Oral Cavity. J Clin Microbiol. 43, 5721-5732 (2005).
  2. Belkaid, Y., Naik, S. Compartmentalized And Systemic Control Of Tissue Immunity By Commensals. Nat Immunol.

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Tags

Gingival Immune CellsImmune Cell IsolationTissue DigestionFlow CytometryCytokine SecretionCollagenase DNaseCell StrainerEx Vivo StimulationPhenotypic CharacterizationFunctional Examination

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