Name: isolation of mouse lung dendritic cells
Uploaded: 2011-11-22T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Mouse Lung Dendritic Cells at JoVE.com

The authors would like to thank to Marilyn Dietrich at the LSU Flow Cytometry Core Facility for her help with the cell sorting and Peter Mottram for his assistance with the microphotographs. This work was funded by the Flight Attendant Medical Research Institute, the LSU-Competitive Research Program Award, and the NIH/NIAID Grants P20 RR020159 and R03AI081171.

1. Lung perfusion and single cell suspension
<ol>
 <li>Euthanize the mouse with an intraperitoneal injection of ketamine/xylazine anesthetic mixture (in mg/mouse ketamine 1.8, xylazine 0.19) and exsanguination via the femoral vein</li>
 <li>Expose the thoracic cavity by cutting and gently pulling back the outer skin of the peritoneum. Proceed to open the diaphragm by cutting the rib cage to expose both the heart and lungs.</li>
 <li>Perfuse gently the lungs using a 5 ml syringe filled with EDTA-HBSS (1mM EDTA in calciu...

<ol>
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Isolation of pulmonary mouse DC is an important technique for the study of a wide range of respiratory stimuli. The process of obtaining these cells includes critical steps that prevent loss of cells as well as cell viability and purity. Perfusing the lung before collection will help to eliminate any peripheral cells as well as reduce contaminant erythrocytes. The use of the automated dissociation can be advanteogus when a large number of lungs are handled, otherwise you can opt for the alternative protocol, keeping in m...

<table width="200" border="1">
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>ACK lysing buffer</td>
 <td>Invitrogen</td>
 <td>D6-0005DG</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-mouse CD11c (HL3)</td>
 <td>BD Pharmingen</td>
 <td>5580979</td>
 <td>PE-Cy7 conjugated</td>
 </tr>
 <tr>
 <td>Anti-mouse I-A/I-E (269)</td>
 <td>BD-Pharmingen</td>
 <td>553623</td>
 <td>FITC conjugated </td>
 </tr>
 <tr>
 <td>Collangenase Type 1A</td>
 <td>Sigma</td>
 <td>9891-500MG</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cell strainers</td>
 <td>BD Falcon</td>
 <td>352340, 352360</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CD11c (N418) Microbeads</td>
 <td>Miltenyi</td>
 <td>130-052-001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DNase I</td>
 <td>Sigma</td>
 <td>D5025-150KU</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hank&rsquo;s Balanced Salt solution</td>
 <td>Invitrogen</td>
 <td>14170</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hepes buffer solution</td>
 <td>Invitrogen</td>
 <td>15630</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Petri dishes 60 mm</td>
 <td>BD Falcon</td>
 <td>351016</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>GentleMACS&trade; C tubes</td>
 <td>Miltenyi</td>
 <td>130-093-237</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Gentle MACS dissociator</td>
 <td>Miltentyi</td>
 <td>130-093-235</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>AutoMACS-Pro&trade;</td>
 <td>Miltenyi</td>
 <td>130-092-545</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FASCS Aria</td>
 <td>BD</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
</table>

isolation of mouse lung dendritic cells

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 respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

Watch this Scientific Journal Video about Isolation of Mouse Lung Dendritic Cells at JoVE.com

Isolation of Mouse Lung Dendritic Cells

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

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 ...

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