Name: Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies (Video) | JoVE Journal
Uploaded: 2012-12-26T12:00:00.000+00:00
Duration: 14 min 48 s
Description: Learn flow cytometric isolation of primary murine type II alveolar epithelial cells for respiratory research. This method yields high purity and viability for functional and molecular studies of lung biology.

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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. Note: Do not perform cervical dislocation as this will injure the trachea so that following steps of the protocol, such as installation of the lung with liquid, cannot be performed successfully. Ensure loss of nociceptive reflexes. </li>
	<li>Spray the mouse with ethanol and make a long cut along the ventral midline of the body. Pull the ventral fur and skin and subsequently also carefully cut and remove the peritoneum.</li>
	<li>Exsanguinate the mouse by cutting the left and right jugular vein (Vena jugularis) as well as the renal artery (Arteria renalis). Remove flowing blood with tissue.</li>
	<li>Carefully puncture and then remove the diaphragm to expose the heart and lung by cutting away the ribs. Take special care not to injure the lung.</li>
	<li>With a 26G cannula and a 10 ml syringe filled with cold phosphate buffered saline (PBS), puncture the right ventricle of the heart and perfuse the lung with PBS until free of blood.</li>
	<li>Cut and remove the salivary glands to expose the trachea. Also carefully cut the muscle surrounding the trachea.</li>
	<li>Insert a 22G indwelling cannula into the trachea, remove the needle and push the plastic catheter towards the lung. Fix the catheter by tying a small piece of yarn around trachea and catheter.</li>
</ol>
2. Enzymatic Digestion of the Lung Tissue
If desired, a bronchoalveolar lavage fluid sample can be prepared by flushing the lungs through the catheter inserted in the trachea with PBS or medium before the next step.
<ol>
	<li>Using a 2 ml syringe, carefully instill a maximal volume of 2 ml of dispase (from the 4 ml aliquot) into the lung through the catheter so that all lobes are fully expanded. Exchange the syringe with a 1 ml syringe containing 0.5 ml of the liquefied agarose and also instill into the lung.</li>
	<li>Leave the syringe on the catheter to prevent back-flow of the agarose and immediately cover the lung with laboratory tissue paper and ice. Let the agarose gel for a couple of minutes.</li>
	<li>Remove the tissue paper, ice, syringe and catheter, cut the trachea and excise the lung, heart and thymus from the chest. Then rinse the lung in a dish with PBS and remove the heart, thymus and remaining trachea.</li>
	<li>Put the lung into the remaining 2 ml of dispase and incubate at room temperature for 45 min.</li>
</ol>
3. Preparation of the Lung Cell Suspension
<ol>
	<li>Remove the lung from the dispase and transfer it into a dish containing 7 ml of anti-CD16/32 antibody (1 &mu;g/ml final concentration) in DMEM medium and 100 &mu;l DNase.</li>
	<li>Using forceps, completely disintegrate the lung tissue by pulling it apart and incubate for 10 min at room temperature while gently rocking on a rocker set to 200 rpm. If desired, the cell suspension can then be stored at 4 &deg;C until the next step.</li>
	<li>Filter the cell suspension through nylon meshes first with 100 &mu;m and subsequently with 70 &mu;m, 48 &mu;m and 30 &mu;m pores. Take care to rinse each mesh as well as the tubes and dishes thoroughly with DMEM to minimize loss of cells and maximize yield. If you are pooling samples, this can be achieved here by subsequently passing cells suspensions from mice of one group through the same filter. Renew the filter in case of clogging.</li>
	<li>Depending on the volume, transfer the filtrate to one or more 50 ml tubes and centrifuge for 15 min at 160 x g and 4 &deg;C. Remove the supernatant.</li>
	<li>Resuspend the cell pellet in 2 ml erythrocyte lysis buffer (0.15 M NH4Cl, 0.01 M KHCO3, 0.1 mM EDTA, pH 7.2) and quickly terminate lysis by addition of 13 ml of DMEM medium. Transfer the solution to a 15 ml tube and centrifuge for 12 min at 160 x g and 4 &deg;C.</li>
</ol>
4. Antibody Staining for Flow Cytometric Cell-sorting
<ol>
	<li>Resuspend the cells in 3 ml primary antibody cocktail, prepared in DMEM medium at dilutions suitable for flow cytometry. (Antibodies were previously titrated for optimal working dilutions by staining 1 x 106 splenocytes in a volume of 100 &mu;l. Use 3 ml of the primary antibody cocktail containing the optimal concentrations for each of the used antibodies for cells pooled from up to five mice. If more mice are pooled to one sample, adjust the volume).</li>
	<li>For staining, incubate the cells for 10 min in the dark at 4 &deg;C.</li>
	<li>Wash the cells by filling the 15 ml tube with DMEM medium and centrifugation for 12 min at 160 x g and 4 &deg;C.</li>
	<li>In case uncoupled or biotinylated antibodies were used in 4.1: Remove the supernatant and resuspend the cells in 2 - 3 ml secondary antibody cocktail. Stain for 10 min at 4 &deg;C in the dark.</li>
	<li>Wash the cells through filling the tube with DMEM and centrifugation for 12 min at 160 x g and 4 &deg;C.</li>
	<li>Resuspend the cells in 1 ml of DMEM and pre-filter through a 50 &mu;m filter into a tube for cell-sorting. Optional: Count the cells to obtain the total cell number in the lung cell suspension.</li>
</ol>
5. Cell-sorting
<ol>
	<li>Mix the cells by vortexing prior to sorting. Use a 100 &mu;m nozzle for cell-sorting of AECII. Sheath fluid pressure as well as laser emission and detection wavelengths depend on the instrument used for flow cytometric cell-sorting as well as the fluorochromes used in the antibody staining procedure.</li>
	<li>Gate on the SSChigh cells that are negative for the fluorochromes used for staining and sort into a tube containing DMEM. Use forward scatter height (FSC-H) vs. area (FSC-A) and sideward scatter height (SSC-H) vs. area (SSC-A) windows to exclude any doublets or cell aggregates (see details of the gating strategy in Figure 1a).</li>
	<li>In order to collect the sorted cells centrifuge for 20 min at 280 x g and 4 &deg;C.</li>
	<li>Resuspend the cells in culture-medium or buffer as required for subsequent analysis.</li>
</ol>

<ol><li>Fehrenbach, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alveolar+epithelial+type+II+cell%3A+defender+of+the+alveolus+revisited%5BTitle%5D)+AND+%22Respir.+Res%22%5BJournal%5D)">Alveolar epithelial type II cell: defender of the alveolus revisited.</a> Respir. Res. 2, 33-46 (2001).</li>
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<li>Gereke, M., Jung, S., Buer, J., Bruder, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alveolar+type+II+epithelial+cells+present+antigen+to+CD4%28%2B%29+T+cells+and+induce+Foxp3%28%2B%29+regulatory+T+cells%5BTitle%5D)+AND+%22Am.+J.+Respir.+Crit+Care+Med%22%5BJournal%5D)">Alveolar type II epithelial cells present antigen to CD4(+) T cells and induce Foxp3(+) regulatory T cells.</a> Am. J. Respir. Crit Care Med. 179, 344-355 (2009).</li>
<li>Gribar, S. C., Richardson, W. M., Sodhi, C. P., Hackam, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((No+longer+an+innocent+bystander%3A+epithelial+toll-like+receptor+signaling+in+the+development+of+mucosal+inflammation%5BTitle%5D)+AND+%22Mol.+Med%22%5BJournal%5D)">No longer an innocent bystander: epithelial toll-like receptor signaling in the development of mucosal inflammation.</a> Mol. Med. 14, 645-659 (2008).</li>
<li>Herold, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alveolar+epithelial+cells+direct+monocyte+transepithelial+migration+upon+influenza+virus+infection%3A+impact+of+chemokines+and+adhesion+molecules%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">Alveolar epithelial cells direct monocyte transepithelial migration upon influenza virus infection: impact of chemokines and adhesion molecules.</a> J. Immunol. 177, 1817-1824 (2006).</li>
<li>Knight, D. A., Holgate, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+airway+epithelium%3A+structural+and+functional+properties+in+health+and+disease%5BTitle%5D)+AND+%22Respirology%22%5BJournal%5D)">The airway epithelium: structural and functional properties in health and disease.</a> Respirology. 8, 432-446 (2003).</li>
<li>Schmiedl, A., Kerber-Momot, T., Munder, A., Pabst, R., Tschernig, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacterial+distribution+in+lung+parenchyma+early+after+pulmonary+infection+with+Pseudomonas+aeruginosa%5BTitle%5D)+AND+%22Cell+Tissue+Res%22%5BJournal%5D)">Bacterial distribution in lung parenchyma early after pulmonary infection with Pseudomonas aeruginosa.</a> Cell Tissue Res. 342, 67-73 (2010).</li>
<li>Loveday, E. K., Svinti, V., Diederich, S., Pasick, J., Jean, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Temporal-+and+Strain-Specific+Host+MicroRNA+Molecular+Signatures+Associated+with+Swine-Origin+H1N1+and+Avian-Origin+H7N7+Influenza+A+Virus+Infection%5BTitle%5D)+AND+%22J.+Virol%22%5BJournal%5D)">Temporal- and Strain-Specific Host MicroRNA Molecular Signatures Associated with Swine-Origin H1N1 and Avian-Origin H7N7 Influenza A Virus Infection.</a> J. Virol. 86, 6109-6122 (2012).</li>
<li>Marriott, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interleukin-1beta+regulates+CXCL8+release+and+influences+disease+outcome+in+response+to+Streptococcus+pneumoniae%2C+defining+intercellular+cooperation+between+pulmonary+epithelial+cells+and+macrophages%5BTitle%5D)+AND+%22Infect.+Immun%22%5BJournal%5D)">Interleukin-1beta regulates CXCL8 release and influences disease outcome in response to Streptococcus pneumoniae, defining intercellular cooperation between pulmonary epithelial cells and macrophages.</a> Infect. Immun. 80, 1140-1149 (2012).</li>
<li>Mata, M., Morcillo, E., Gimeno, C., Cortijo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((N-acetyl-L-cysteine+%28NAC%29+inhibit+mucin+synthesis+and+pro-inflammatory+mediators+in+alveolar+type+II+epithelial+cells+infected+with+influenza+virus+A+and+B+and+with+respiratory+syncytial+virus+%28RSV%29%5BTitle%5D)+AND+%22Biochem.+Pharmacol%22%5BJournal%5D)">N-acetyl-L-cysteine (NAC) inhibit mucin synthesis and pro-inflammatory mediators in alveolar type II epithelial cells infected with influenza virus A and B and with respiratory syncytial virus (RSV).</a> Biochem. Pharmacol. 82, 548-555 (2011).</li>
<li>Zarbock, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+surfactant+protein+C+mutation+A116D+alters+cellular+processing%2C+stress+tolerance%2C+surfactant+lipid+composition%2C+and+immune+cell+activation%5BTitle%5D)+AND+%22BMC.+Pulm.+Med%22%5BJournal%5D)">The surfactant protein C mutation A116D alters cellular processing, stress tolerance, surfactant lipid composition, and immune cell activation.</a> BMC. Pulm. Med. 12, 15(2012).</li>
<li>Taubenberger, J. K., Morens, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+pathology+of+influenza+virus+infections%5BTitle%5D)+AND+%22Annu.+Rev.+Pathol%22%5BJournal%5D)">The pathology of influenza virus infections.</a> Annu. Rev. Pathol. 3, 499-522 (2008).</li>
<li>Dobbs, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+culture+of+alveolar+type+II+cells%5BTitle%5D)+AND+%22Am.+J.+Physiol%22%5BJournal%5D)">Isolation and culture of alveolar type II cells.</a> Am. J. Physiol. 258, L134-L147 (1990).</li>
<li>Corti, M., Brody, A. R., Harrison, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+primary+culture+of+murine+alveolar+type+II+cells%5BTitle%5D)+AND+%22Am.+J.+Respir.+Cell+Mol.+Biol%22%5BJournal%5D)">Isolation and primary culture of murine alveolar type II cells.</a> Am. J. Respir. Cell Mol. Biol. 14, 309-315 (1996).</li>
<li>Beers, M. F., Kim, C. Y., Dodia, C., Fisher, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Localization%2C+synthesis%2C+and+processing+of+surfactant+protein+SP-C+in+rat+lung+analyzed+by+epitope-specific+antipeptide+antibodies%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">Localization, synthesis, and processing of surfactant protein SP-C in rat lung analyzed by epitope-specific antipeptide antibodies.</a> J. Biol. Chem. 269, 20318-20328 (1994).</li>
<li>Phelps, D. S., Floros, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Localization+of+pulmonary+surfactant+proteins+using+immunohistochemistry+and+tissue+in+situ+hybridization%5BTitle%5D)+AND+%22Exp.+Lung+Res%22%5BJournal%5D)">Localization of pulmonary surfactant proteins using immunohistochemistry and tissue in situ hybridization.</a> Exp. Lung Res. 17, 985-995 (1991).</li>
<li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differentiated+human+alveolar+type+II+cells+secrete+antiviral+IL-29+%28IFN-lambda+1%29+in+response+to+influenza+A+infection%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">Differentiated human alveolar type II cells secrete antiviral IL-29 (IFN-lambda 1) in response to influenza A infection.</a> J. Immunol. 182, 1296-1304 (2009).</li>
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No conflicts of interest declared.

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><tbody><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 &amp;deg;C</td></tr><tr><td>Biozym Plaque Agarose</td><td>Biozym</td><td>840101</td><td>1% w/v in H&lt;sub&gt;2&lt;/sub&gt;O</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 &amp;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 &amp;mu;m, 75 &amp;mu;m) </td><td>BD Falcon</td><td>352360, 352350</td><td></td></tr><tr><td>nylon mesh(48 &amp;mu;m, 30 &amp;mu;m) </td><td>B&amp;#252;ckmann GmbH</td><td>03-48/26-1020, 03-30/18-108</td><td></td></tr><tr><td>CellTrics 50 &amp;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 colspan="4">&lt;p class=&quot;jove_content&quot;&gt;Antibodies coupled to alternative fluorochromes can be used, depending on the flow cytometer and lasers available.&lt;/p&gt; </td></tr></tbody></table>

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

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 ± 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 in the A...

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 ...

The overall aim of this procedure is to isolate pure and viable primary neuron type two alveolar epithelial cells for subsequent functional and molecular analyses. First, the lung and trachea of a sacrificed mouse are instilled with a solution of DYS displays and then agarose. The lung is then removed after the dispa is allowed to digest the lung.The tissue is manually disintegrated. The resulting suspension is filtered through a series of meshes of decreasing pore size. Then stained and type two alveolar epithelial cells are isolated by flow cytometric negative selection.Ultimately, the isolated type two alveolar epithelial cells can be used to perform functional and molecular assays to study their role in respiratory infection and pary autoimmune diseases. This method can help answer key questions in the field of pulmonary immune relation, especially in the context of autoimmune mediad lung diseases and viral infections. Visual demonstration of this method is critical as the organ and cell suspension preparation are difficult to learn.The overall success of this protocol depends on several important details during these steps For each mouse in the experiment, prewarm a 15 millili

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Learn flow cytometric isolation of primary murine type II alveolar epithelial cells for respiratory research. This method yields high purity and viability for functional and molecular studies of lung biology.

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

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