Name: flow cytometric isolation of primary murine type ii alveolar epithelial cells for functional and molecular studies
Uploaded: 2012-12-26T12:00:00
Description: Watch this Scientific Journal Video about Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies at JoVE.com

We would like to thank M. H&ouml;xter for technical assistance in sorting primary murine AECII from biosafety level 2 samples.
This work was supported by grants from the German Research Foundation (DFG) to DB (SFB587, TP B12 and BR2221/1-1) and a stipend from the Hannover Biomedical Research School (DFG GSC 108) to AA. DB is supported by the President&acute;s Initiative and Networking Fund of the Helmholtz Association of German Research Centers (HGF) under contract number W2/W3-029.

Details regarding required reagents and materials are listed in the table at the end of the protocol below. Before starting work, prepare 15 ml tubes (one per mouse) containing 4 ml of dispase and pre-warm them to 37 &deg;C in a water bath. In a heating block, shortly heat small aliquots of 1 % low-melt agarose (in water) to 95 &deg;C until liquefied and subsequently cool to 45 &deg;C until use.
1. Preparation of the Mouse Lung
<ol>
	<li>Sacrifice the mouse by CO2 asphyxiation. <s...

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Our protocol for the isolation of murine AECII by flow cytometry offers a rapid way of accessing primary cells from the mouse lung for a whole range of functional and molecular studies. The described procedure yields highly viable and pure populations of AECII that are sufficient in number for direct subsequent analyses, such as RNA isolation (see Figure 2b) and transcriptome studies. For functional applications, it is also possible to culture the isolated cells, allowing e.g. the generation of ...

<table border="1">
 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>indwelling cannula Introcan 22G</td>
 <td>Braun</td>
 <td>REF 4252098B</td>
 <td></td>
 </tr>
 <tr>
 <td>Dispase, 100 ml(5000 caseinolytic units)</td>
 <td>BD Biosciences</td>
 <td>354235</td>
 <td>aliquot to 4 ml in 15 ml tubes, store at -20 &deg;C</td>
 </tr>
 <tr>
 <td>Biozym Plaque Agarose</td>
 <td>Biozym</td>
 <td>840101</td>
 <td>1% w/v in H2O</td>
 </tr>
 <tr>
 <td>Deoxyribonuclease I from bovine pancreas, 2000 Kunitz units/vial</td>
 <td>Sigma-Aldrich</td>
 <td>D4263</td>
 <td>freshly dissolve content of 1 vial in 300 &mu;l DMEM</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Gibco</td>
 <td>22320-022</td>
 <td>used as provided by manufacturer (Low Glucose, Pyruvate, HEPES)</td>
 </tr>
 <tr>
 <td>cell strainers (100 &mu;m, 75 &mu;m) </td>
 <td>BD Falcon</td>
 <td>352360, 352350</td>
 <td></td>
 </tr>
 <tr>
 <td>nylon mesh(48 &mu;m, 30 &mu;m) </td>
 <td>B&uuml;ckmann GmbH</td>
 <td>03-48/26-1020, 03-30/18-108</td>
 <td></td>
 </tr>
 <tr>
 <td>CellTrics 50 &mu;m filter</td>
 <td>PARTEC</td>
 <td>04-0042-2317</td>
 <td></td>
 </tr>
 <tr>
 <td>anti-mouse CD16/CD32</td>
 <td>BioLegend</td>
 <td>101302</td>
 <td>clone 93; purified</td>
 </tr>
 <tr>
 <td>anti-mouse F4/80</td>
 <td>BioLegend</td>
 <td>123116</td>
 <td>clone BM8; APC coupled</td>
 </tr>
 <tr>
 <td>anti-mouse CD11b</td>
 <td>BioLegend</td>
 <td>101208</td>
 <td>clone M1/70; PE coupled</td>
 </tr>
 <tr>
 <td>anti-mouse CD11c</td>
 <td>BioLegend</td>
 <td>117310</td>
 <td>clone N418; APC coupled</td>
 </tr>
 <tr>
 <td>anti-mouse CD45</td>
 <td>BioLegend</td>
 <td>103102</td>
 <td>clone 30-F11; purified</td>
 </tr>
 <tr>
 <td>anti-mouse CD19</td>
 <td>eBioscience</td>
 <td>12-0193-83</td>
 <td>eBio 1D3; PE coupled</td>
 </tr>
 <tr>
 <td>polyclonal
 goat anti-rat IgG</td>
 <td>BD 
 Pharmingen</td>
 <td>550767</td>
 <td>polyclonal, PE coupled</td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>Antibodies coupled to alternative fluorochromes can be used, depending on the flow cytometer and lasers available.
</td>
 </tr>
 </tbody>
</table>

flow cytometric isolation of primary murine type ii alveolar epithelial cells for functional and molecular studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

When sorting lung cell suspensions isolated from healthy mice, the AECII gate will typically account for about 42 &plusmn; 10 % of all events. This percentage can be noticeably lower when mice with respiratory conditions such as a viral infection are used, as the initial cell suspension will contain a considerably higher proportion of lymphocytes and other immune cells recruited to the airways. For AECII isolated from IAV infected lungs on day 3 following infection we have observed a reduction of the frequency of cells i...

Watch this Scientific Journal Video about Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies at JoVE.com

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

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