Name: isolation and characterization of dendritic cells and macrophages from the mouse intestine
Uploaded: 2012-05-21T13:00:00
Description: Watch this Scientific Journal Video about Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine at JoVE.com

Ringraziamo Aaron Rae (Emory University Dipartimento di Pediatria e Healthcare bambini di Core flusso Atlanta) per l&#39;ordinamento delle cellule. Questo lavoro è stato supportato da sovvenzioni NIH AA01787001, un Career Development Award dal morbo di Crohn e Colite Foundation of America, e della ricerca uno Emory-Egleston Children Center di sementi sovvenzione TLD

1. Dissezione e la dissociazione di cellule epiteliali intestinali Preparazione dei reagenti e attrezzature: <ul><li> Caldo Ca 2 + / Mg 2 +-libera PBS (CMF PBS) a temperatura ambiente. </li><li> Caldo Ca 2 + / Mg 2 +-libere HBSS con 5% FBS (CMF HBSS / FBS) e 2 mM EDTA a temperatura ambiente. </li><li> Warm Agitatore orbitale a 37 ° C. </li></ul> Nota: Procedura 1,1 a 1,7 devono essere eseguite più rapida...

<ol>
	<li>Maloy, K. J., Powrie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+homeostasis+and+its+breakdown+in+inflammatory+bowel+disease.">Intestinal homeostasis and its breakdown in inflammatory bowel disease.</a> Nature. 474, 298-306 (2011).</li><li>Nagler-Anderson, C., Terhoust, C., Bhan, A. K., Podolsky, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+antigen+presentation+and+the+control+of+tolerance+and+immunity.">Mucosal antigen presentation and the control of tolerance and immunity.</a> Trends Immunol. 22, 120-122 (2001).</li><li>Abraham, C., Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+the+host+innate+immune+system+and+microbes+in+inflammatory+bowel+disease.">Interactions between the host innate immune system and microbes in inflammatory bowel disease.</a> Gastroenterology. 140, 1729-1737 (2011).</li><li>Macdonald, T. T., Monteleone, I., Fantini, M. C., Monteleone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regula+tine.">Regula tine.</a> Gastroenterology. 140, 1768-1775 (2011).</li><li>Rescigno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+dendritic+cells.">Intestinal dendritic cells.</a> Adv. Immunol. 107, 109-138 (2010).</li><li>Platt, A. M., Bain, C. C., Bordon, Y., Sester, D. P., Mowat, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+independent+subset+of+TLR+expressing+CCR2-dependent+macrophages+promotes+colonic+inflammation.">An independent subset of TLR expressing CCR2-dependent macrophages promotes colonic inflammation.</a> J. Immunol. 184, 6843-6854 (2010).</li><li>Coombes, J. L., Powrie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+in+intestinal+immune+regulation.">Dendritic cells in intestinal immune regulation.</a> Nat. Rev. Immunol. 8, 435-446 (2008).</li><li>Kelsall, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+understanding+the+phenotype+and+function+of+intestinal+dendritic+cells+and+macrophages.">Recent progress in understanding the phenotype and function of intestinal dendritic cells and macrophages.</a> Mucosal Immunol. 1, 460-469 (2008).</li><li>Pulendran, B., Tang, H., Denning, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Division+of+labor,+plasticity,+and+crosstalk+between+dendritic+cell+subsets.">Division of labor, plasticity, and crosstalk between dendritic cell subsets.</a> Curr. Opin. Immunol. 20, 61-67 (2008).</li><li>Denning, T. L., Wang, Y. C., Patel, S. R., Williams, I. R., Pulendran, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lamina+propria+macrophages+and+dendritic+cells+differentially+induce+regulatory+and+interleukin+17-producing+T+cell+responses.">Lamina propria macrophages and dendritic cells differentially induce regulatory and interleukin 17-producing T cell responses.</a> Nat. Immunol. 8, 1086-1094 (2007).</li><li>Niess, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CX3CR1-mediated+dendritic+cell+access+to+the+intestinal+lumen+and+bacterial+clearance.">CX3CR1-mediated dendritic cell access to the intestinal lumen and bacterial clearance.</a> Science. 307, 254-258 (2005).</li><li>Milling, S. W., Cousins, L., MacPherson, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+DCs+interact+with+intestinal+antigens.">How do DCs interact with intestinal antigens.</a> Trends Immunol. 26, 349-352 (2005).</li><li>Bilsborough, J., Viney, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+dendritic+cells+play+a+role+in+immunity,+tolerance,+and+disease.">Gastrointestinal dendritic cells play a role in immunity, tolerance, and disease.</a> Gastroenterology. 127, 300-309 (2004).</li><li>Stagg, A. J., Hart, A. L., Knight, S. C., Kamm, 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=The+dendritic+cell:+its+role+in+intestinal+inflammation+and+relationship+with+gut+bacteria.">The dendritic cell: its role in intestinal inflammation and relationship with gut bacteria.</a> Gut. 52, 1522-1529 (2003).</li><li>Medina-Contreras, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CX3CR1+regulates+intestinal+macrophage+homeostasis,+bacterial+translocation,+and+colitogenic+Th17+responses+in+mice.">CX3CR1 regulates intestinal macrophage homeostasis, bacterial translocation, and colitogenic Th17 responses in mice.</a> J. Clin. Invest. 121, 4787-4795 (2011).</li><li>Denning, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Specializations+of+Intestinal+Dendritic+Cell+and+Macrophage+Subsets+That+Control+Th17+and+Regulatory+T+Cell+Responses+Are+Dependent+on+the+T+Cell/APC+Ratio,+Source+of+Mouse+Strain,+and+Regional+Localization.">Functional Specializations of Intestinal Dendritic Cell and Macrophage Subsets That Control Th17 and Regulatory T Cell Responses Are Dependent on the T Cell/APC Ratio, Source of Mouse Strain, and Regional Localization.</a> J. Immunol. , 187-733 (2011).</li><li>Kim, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Nod2+sensor+promotes+intestinal+pathogen+eradication+via+the+chemokine+CCL2-dependent+recruitment+of+inflammatory+monocytes.">The Nod2 sensor promotes intestinal pathogen eradication via the chemokine CCL2-dependent recruitment of inflammatory monocytes.</a> Immunity. 34, 769-780 (2011).</li><li>Schulz, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+CD103+,+but+not+CX3CR1+,+antigen+sampling+cells+migrate+in+lymph+and+serve+classical+dendritic+cell+functions.">Intestinal CD103+, but not CX3CR1+, antigen sampling cells migrate in lymph and serve classical dendritic cell functions.</a> J. Exp. Med. 206, 3101-3114 (2009).</li><li>Jaensson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+intestinal+CD103++dendritic+cells+display+unique+functional+properties+that+are+conserved+between+mice+and+humans.">Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans.</a> J. Exp. Med. 205, 2139-2149 (2008).</li><li>Uematsu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+humoral+and+cellular+gut+immunity+by+lamina+propria+dendritic+cells+expressing+Toll-like+receptor+5.">Regulation of humoral and cellular gut immunity by lamina propria dendritic cells expressing Toll-like receptor 5.</a> Nat. Immunol. 9, 769-776 (2008).</li><li>Schenk, M., Bouchon, A., Seibold, F., Mueller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TREM-1--expressing+intestinal+macrophages+crucially+amplify+chronic+inflammation+in+experimental+colitis+and+inflammatory+bowel+diseases.">TREM-1--expressing intestinal macrophages crucially amplify chronic inflammation in experimental colitis and inflammatory bowel diseases.</a> J. Clin. Invest. 117, 3097-3106 (2007).</li><li>Sun, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+intestine+lamina+propria+dendritic+cells+promote+de+novo+generation+of+Foxp3+T+reg+cells+via+retinoic+acid.">Small intestine lamina propria dendritic cells promote de novo generation of Foxp3 T reg cells via retinoic acid.</a> J. Exp. Med. 204, 1775-1785 (2007).</li><li>Kamada, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormally+differentiated+subsets+of+intestinal+macrophage+play+a+key+role+in+Th1-dominant+chronic+colitis+through+excess+production+of+IL-12+and+IL-23+in+response+to+bacteria.">Abnormally differentiated subsets of intestinal macrophage play a key role in Th1-dominant chronic colitis through excess production of IL-12 and IL-23 in response to bacteria.</a> J. Immunol. 175, 6900-6908 (2005).</li><li>Denning, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++Th+cells+resembling+regulatory+T+cells+that+inhibit+chronic+colitis+differentiate+in+the+absence+of+interactions+between+CD4+and+class+II+MHC.">CD4+ Th cells resembling regulatory T cells that inhibit chronic colitis differentiate in the absence of interactions between CD4 and class II MHC.</a> J. Immunol. 171, 2279-2286 (2003).</li></ol>

<img src="/files/ftp_upload/4040/4040fig3.jpg" alt="Figura 3"/> Figura 3. Fattori importanti per l&#39;ottimizzazione della resa delle cellule e l&#39;espressione dell&#39;antigene di superficie. Resa cellulare e l&#39;espressione dell&#39;antigene di superficie sono direttamente interessati dalla durata della digestione dei tessuti, le caratteristiche specifiche del collagenasi, il grado di macinazione dei tessuti e la presenza o l&#39;assenza di infiammazione, che possano pregiudicare l&#39;integ...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> 1X PBS, Ca 2 + - 2 + e Mg-free </td><td></td><td></td><td></td></tr><tr><td> Sale di Hank soluzione equilibrata (HBSS) con rosso fenolo </td><td> Fisher Scientific </td><td> SH3001603 </td><td></td></tr><tr><td> Bicarbonato di sodio </td><td> Sigma </td><td> S6014 </td><td></td></tr><tr><td> HEPES 1M in 0,85% NaCl </td><td> Lonza </td><td> 17-737E </td><td></td></tr><tr><td> Siero bovino fetale (FBS) </td><td> Atlanta biologici </td><td> S11150H </td><td> Inattivato al calore </td></tr><tr><td> 0,5 M EDTA (pH 8,0) </td><td> Cellgro </td><td> 46-034-CI </td><td></td></tr><tr><td> Collagenase di tipo VIII </td><td> Sigma </td><td> C2139 </td><td></td></tr><tr><td> DNasi I </td><td> Roche </td><td> 14785000 </td><td> Soluzione madre: 100mg/ml </td></tr><tr><td> LIVE / DEAD fissabile Aqua Kit Dead Cell Stain per 405 nm di eccitazione </td><td> Invitrogen </td><td> L34957 </td><td> Utilizzare a 1:1000 </td></tr><tr><td> CD45-PerCP mAb (30F11) </td><td> BD </td><td> 557235 </td><td> Utilizzare a 1:100 </td></tr><tr><td> CD103-PE mAb (M290) </td><td> BD </td><td> 557495 </td><td> Utilizzare a 1:100 </td></tr><tr><td> FcyRIII / II mAb (2.4G2) </td><td> BD </td><td> 553141 </td><td> Utilizzare a 1:200 </td></tr><tr><td> CD11c-APC mAb (N418) </td><td> eBioscience </td><td> 17-0114-82 </td><td> Utilizzare a 1:100 </td></tr><tr><td> MHC-II (I-Ab)-Alexa Fluor 700 mAb </td><td> eBioscience </td><td> 56-5321-82 </td><td> Utilizzare a 1:100 </td></tr><tr><td> CD11b-eFluor 450mAb (M1/70) </td><td> eBioscience </td><td> 48-0112-82 </td><td> Utilizzare a 1:200 </td></tr><tr><td> F4/80-PE-Cy7 mAb (BM8) </td><td> eBioscience </td><td> 25-4801-82 </td><td></td></tr><tr><td> CD11b microsfere </td><td> Miltenyi Biotec </td><td> 130-049-601 </td><td></td></tr><tr><td> CD11c microsfere </td><td> Miltenyi Biotec </td><td> 130-052-001 </td><td></td></tr><tr><td> 50 ml provette coniche </td><td> BD Falcon </td><td> 352098 </td><td></td></tr><tr><td> Singola mesh filo </td><td> Chefmate </td><td></td><td></td></tr><tr><td> Piccola pesano barca </td><td> Fisher Scientific </td><td> 08-732-116 </td><td></td></tr><tr><td> Filtro da 100 micron delle cellule </td><td> BD Falcon </td><td> 352360 </td><td></td></tr><tr><td> 40 micron filtro cella </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> 5 ml in polistirolo a fondo tondo tubi </td><td> BD Falcon </td><td> 352235 </td><td> Utilizzare a 1:100 </td></tr><tr><td> MaxQ 4450 agitatore orbitale da banco </td><td> Thermo Scientific </td><td></td><td></td></tr><tr><td> LS MACS colonna </td><td> Miltenyi Biotec </td><td> 130-042-401 </td><td></td></tr><tr><td> LSR II </td><td> BD </td><td></td><td></td></tr><tr><td> FACSAria II </td><td> BD </td><td></td><td></td></tr></tbody></table>

isolation and characterization of dendritic cells and macrophages from the mouse intestine

All&#39;interno l&#39;intestino risiedono popolazioni uniche di cellule immunitarie innate e adattiva che sono coinvolti nella promozione della tolleranza verso la flora commensale e gli antigeni alimentari, mentre in concomitanza rimanendo in bilico per montare le risposte infiammatorie verso invasive 1,2 patogeni. Cellule presentanti l&#39;antigene, in particolare DCS e macrofagi, giocano un ruolo critico nel mantenimento della omeostasi immunitaria intestinale attraverso la loro capacità di percepire e rispondere adeguatamente al microbiota 3-14. Isolamento efficiente di DC intestinali e macrofagi è un passo fondamentale nel caratterizzare il fenotipo e la funzione di queste cellule. Mentre molti metodi efficaci per isolare cellule immunitarie intestinali, compresi DC e macrofagi, sono stati descritti 6,10,15-24, contare su lunghe molte volte digestioni che possono influenzare negativamente l&#39;espressione sulla superficie cellulare antigene, la vitalità cellulare, e / o quantità di cellule. Qui, si dettaglio una metodologia per l&#39;isolamento rapido di un gran numero di viablelettronici, DCS intestinali e macrofagi. Caratterizzazione fenotipica delle DC intestinali e macrofagi viene effettuata direttamente colorazione cellule intestinali isolate con specifici anticorpi monoclonali marcati a fluorescenza per multi-colore analisi citofluorimetrica. Inoltre, DC altamente puro e popolazioni macrofagi sono isolati per studi funzionali utilizzano CD11c e CD11b magnetico attivati ​​perline di separazione delle cellule seguite da selezione cellulare.

Watch this Scientific Journal Video about Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine at JoVE.com

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Qui, dettaglio una metodologia per l&#39;isolamento rapido delle cellule dendritiche di topo intestinali (DCS) e macrofagi. Caratterizzazione fenotipica di DC intestinali e macrofagi viene eseguita utilizzando multi-color analisi citometrica mentre magnetica arricchimento tallone seguita dalla selezione cellulare viene utilizzato per produrre le popolazioni di elevata purezza per gli studi funzionali.

Isolamento e caratterizzazione di cellule dendritiche ei macrofagi dall&#39;intestino mouse

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Research

JoVE Journal

Immunology and Infection

Isolation of Mouse Lung Dendritic Cells

Isolamento di cellule polmonari mouse dendritiche

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Ricerca

Immunologia e infezioni

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

Isolamento di cellule dendritiche mieloidi e cellule epiteliali di timo umano

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Cellule T Capture batteri da Transinfection da cellule dendritiche

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Isolamento e analisi citofluorimetrica delle cellule immunitarie dal cervello ischemica mouse

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Generazione e identificazione di GM-CSF Derivato alveolo-come macrofagi e dendritiche cellule da midollo osseo di topo

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Caratterizzazione delle derivate da monociti cellule dendritiche umane di Citometria a Flusso Imaging: un confronto tra due protocolli di isolamento dei monociti

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

Caratterizzazione fenotipica dei macrofagi dal rene di ratto mediante citometria di flusso

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Separazione delle cellule fluorescenza-attivato per la purificazione delle cellule dendritiche plasmacitoidi dal midollo osseo di topo

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Generazione di derivate da monociti cellule dendritiche umane da sangue intero

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Isolamento, caratterizzazione e purificazione dei macrofagi dai tessuti colpiti da infiammazione obesità

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...