We thank Aaron Rae (Emory University Department of Pediatrics and Children's Healthcare of Atlanta Flow Core) for cell sorting. This work was supported by NIH grant AA01787001, a Career Development Award from the Crohn's and Colitis Foundation of America, and an Emory-Egleston Children's Research Center seed grant to T.L.D.

1. Dissection and Dissociation of Intestinal Epithelial Cells
Preparation of reagents and equipment:
<ul>
	<li>Warm Ca2+/Mg2+-free PBS (CMF PBS) to room temperature.</li>
	<li>Warm Ca2+/Mg2+-free HBSS with 5% FBS (CMF HBSS/FBS) and 2mM EDTA to room temperature.</li>
	<li>Warm Orbital shaker to 37 &deg;C.</li>
</ul>
Note: Steps 1.1 to 1.7 must be performed as quickly as possible to minimize the extent of cell...

<ol>
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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. 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No conflicts of interest declared.

<img src="/files/ftp_upload/4040/4040fig3.jpg" alt="Figure 3"/> 
Figure 3. Factors important for the optimization of cell yield and surface antigen expression. Cell yield and surface antigen expression are directly affected by the duration of tissue digestion, the specific characteristics of the collagenase, the degree of tissue mincing, and the presence or absence of inflammation, which may affect tissue integrity and cellularity. Prolonged tissue digestion may result in decreased cell viabili...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the Reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>1X PBS, Ca2+- and Mg2+-free</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hank's balanced salt solution (HBSS) with phenol red</td>
 <td>Fisher Scientific</td>
 <td>SH3001603</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium bicarbonate</td>
 <td>Sigma</td>
 <td>S6014</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1M HEPES in 0.85% NaCl</td>
 <td>Lonza</td>
 <td>17-737E</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal bovine serum (FBS)</td>
 <td>Atlanta biologicals</td>
 <td>S11150H</td>
 <td>Heat-inactivated</td>
 </tr>
 <tr>
 <td>0.5M EDTA (pH 8.0)</td>
 <td>Cellgro</td>
 <td>46-034-CI</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Collagenase type VIII </td>
 <td>Sigma</td>
 <td>C2139</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DNase I</td>
 <td>Roche</td>
 <td>14785000</td>
 <td>Stock solution: 100mg/ml</td>
 </tr>
 <tr>
 <td>LIVE/DEAD Fixable Aqua Dead Cell Stain Kit for 405 nm excitation</td>
 <td>Invitrogen</td>
 <td>L34957</td>
 <td>Use at 1:1000</td>
 </tr>
 <tr>
 <td>CD45-PerCP mAb (30F11)</td>
 <td>BD</td>
 <td>557235</td>
 <td>Use at 1:100</td>
 </tr>
 <tr>
 <td>CD103-PE mAb (M290)</td>
 <td>BD</td>
 <td>557495</td>
 <td>Use at 1:100</td>
 </tr>
 <tr>
 <td>Fc&#x03B3;RIII/II mAb (2.4G2)</td>
 <td>BD</td>
 <td>553141</td>
 <td>Use at 1:200</td>
 </tr>
 <tr>
 <td>CD11c-APC mAb (N418)</td>
 <td>eBioscience</td>
 <td>17-0114-82</td>
 <td>Use at 1:100</td>
 </tr>
 <tr>
 <td>MHC-II (I-Ab)-Alexa Fluor 700 mAb </td>
 <td>eBioscience</td>
 <td>56-5321-82</td>
 <td>Use at 1:100</td>
 </tr>
 <tr>
 <td>CD11b-eFluor 450 mAb (M1/70)</td>
 <td>eBioscience</td>
 <td>48-0112-82</td>
 <td>Use at 1:200 </td>
 </tr>
 <tr>
 <td>F4/80-PE-Cy7 mAb (BM8)</td>
 <td>eBioscience</td>
 <td>25-4801-82</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CD11b microbeads</td>
 <td>Miltenyi Biotec</td>
 <td>130-049-601</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CD11c microbeads</td>
 <td>Miltenyi Biotec</td>
 <td>130-052-001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50 mL conical tubes</td>
 <td>BD Falcon</td>
 <td>352098</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Single mesh wire strainer</td>
 <td>Chefmate</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Small weigh boat</td>
 <td>Fisher Scientific</td>
 <td>08-732-116</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>100 &mu;m cell strainer</td>
 <td>BD Falcon</td>
 <td>352360</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>40 &mu;m cell strainer</td>
 <td>BD Falcon</td>
 <td>352340</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>5 mL polystyrene round-bottom tubes</td>
 <td>BD Falcon</td>
 <td>352235</td>
 <td>Use at 1:100</td>
 </tr>
 <tr>
 <td>MaxQ 4450 benchtop orbital shaker</td>
 <td>Thermo Scientific</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>LS MACS column</td>
 <td>Miltenyi Biotec</td>
 <td>130-042-401</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>LSR II</td>
 <td>BD</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FACSAria II</td>
 <td>BD</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

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

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

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

isolation-characterization-dendritic-cells-macrophages-from-mouse

Research

JoVE Journal

Immunology and Infection

38.9K 조회수.  Emory University. 여기서는 자세하게 마우스 장 돌기 세포 (DC가)와 macrophages의 급속한 분리를위한 방법론. 창자 DCS 및 macrophages의 Phenotypic 특성은 세포 정렬 뒤에 자석 구슬 농축은 기능적 연구를위한 매우 순수 인구를 얻을하는 데 사용하는 동안 멀티 컬러 플로우에게 cytometric 분석을 사용하여 수행됩니다.