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>

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<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>
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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 milliliter tube containing four milliliters of DYS bays in a 37 degrees Celsius water bath place, small aliquots of 1%low. Mel Aros in water in a 95 degrees Celsius heating block until liquefied. Then call to 45 degrees Celsius and maintain them at this temperature until they are used next.To prepare the mouse lung spray a mouse that has been sacrificed by CO2 asphyxiation. With ethanol. Make a long cut along the ventral midline of the body.Then pull the ventral far skin and carefully cut and remove the peritoneum. After exsanguinating the mouse, carefully puncture and remove the diaphragm and then cut away the ribs. To expose the heart and lung, take special care not to injure the lung with a 26 gauge cannula and a 10 milliliter syringe filled with cold.PBS puncture the right ventricle of the heart and perfuse the lung with PBS until it is free of blood. Cut and remove the salivary glands to expose the trachea. Also, carefully cut the muscles surrounding the trachea.Next, insert a 22 gauge indwelling cannula into the trachea. Remove the needle and push the plastic catheter towards the lung. Fix the catheter into place by tying a small piece of yarn around the trachea and catheter.If desired, prepare a bronchoalveolar lavage fluid sample by flushing the lungs using the catheter inserted in the trachea with PBS or medium. Store the lavage fluid on ice until further analysis. The single most difficult aspect of this procedure is digestion and tissue disintegration.To ensure the success of this protocol, you have to take care that the complete dispa is filled up in all of the lung lobes and after tissue digestion. You have to disintegrate the tissue Using a two milliliter syringe carefully, still a maximum volume of two milliliters of the dispa prepared earlier into the lung through the catheter so that all lobes are fully expanded. Exchange the syringe for a one milliliter syringe containing 0.5 milliliters of the liquified aros and instill the contents into the lung.Leave the syringe on the catheter to prevent backflow of the aros and immediately cover the lung with laboratory tissue, paper and ice. Let the aero gel for a couple of minutes. Remove the tissue paper, ice syringe, and catheter.Then cut the trachea and excise the lung, heart, and thymus from the chest. Rinse the prepared organs in a dish with PBS. Then cut the heart thymus and remaining trachea from the lung and discard them.Put the lung into the tube containing the remaining two milliliters of displays and incubate it at room temperature for 45 minutes. Alter the incubation to ensure efficient blocking of the FC receptors of phagocytic cells before the staining procedure. Transfer the lung from the displays into a dish containing seven milliliters of one microgram per milliliter, anti CD 1632 antibody and 100 microliters of DNAs in DMEM, using two pairs of forceps completely disintegrate the lung tissue by pulling it apart.Then incubate it for 10 minutes at room temperature on a rocker set to 200 RPM if desired. The cell suspension can then be stored at four degrees Celsius until the next step. Pipette the cell suspension through a series of nylon meshes.First, pour the suspension through a 100 micron filter placed on top of a 50 milliliter tube. If pooling samples, pass the cell suspensions from mice of the same group through the same filter. If the filter clogs, exchange it for a new one.Repeat this process with a 70 micron mesh filter. Take care to rinse each mesh as well as the tubes and dishes thoroughly with DMEM to minimize loss of cells and maximize yield. Next, pull the suspension through a 48 micron mesh filter span onto a plastic or glass ring as shown here.Collect the flow through in a Petri dish. Then repeat this process with a 30 micron mesh. Depending on the volume, transfer the filtrate to one or more 50 milliliters, tubes and centrifuge for 15 minutes of 160 times G at four degrees Celsius.After the spin, remove the supinate and resuspend the cell pellet in two milliliters of erythrocyte lysis buffer. Then quickly terminate lysis by addition of 13 milliliters of DMEM medium. Transfer the solution to a 15 milliliter tube and centrifuge for 12 minutes at 160 times G and four degrees Celsius.Reese has been the cells in three milliliters of a primary antibody cocktail containing antibodies against CD 11 C, CD 11 BF four 80 CD 19, CD 1632, and CD 45. Incubate the cells for 10 minutes in the dark at four degrees Celsius or on ice after the incubation. Wash the cells by filling the tube with DMEM and centrifuge for 12 minutes, a 160 times G and four degrees Celsius.Re suspend the cells in one milliliter of DMEM and pre-filter through a 50 micron filter into a tube for cell sorting. Count the cells to obtain the total cell number in the lung suspension. Use a 100 micro nozzle for cell sourcing of a EC2 immediately before sourcing.Mix the cell suspension by vortexing sheath. Fluid pressure and laser remission and detection wavelengths depend on the instrument used for flow cytometric cell sorting as well as the fluorochromes used in the antibody staining procedure. First, create a sideward scatter area versus fluorescence plot and gate on all sideward scatter area High cells negative for the fluorochromes used for staining.Next, generate sideward scatter area versus forward scatter area and forward scatter height versus forward scatter area and sideward scatter height versus sideward scatter area plots and gate to exclude any doublets or cell aggregates source into a tube containing DMEM. When sourcing lung cell suspensions isolated from healthy mice, the a EC2 gate will typically account for about 42 plus minus 10%of all events. In order to collect the sorted cells centrifuge for 20 minutes at 280 times G and four degrees Celsius.Re suspend the type two alveolar epithelial cells or a EC2 in culture, media or buffer as required for subsequent analysis. Primary murine type two alveolar epithelial cells were isolated by flow cytometric negative selection as described in this video, analysis of the sorted cells is depicted here. As indicated.The purity of the isolated cells is roughly 92 plus minus 5%Ciis spins of sorted A EC2 was stained for the surfactant protein CA phase contrast image is shown on the left and immunofluorescence is shown on the right. Nearly 100%of cells were found to be surfactant protein C positive, thus confirming successful isolation of a pure population. To assess the effects of in vivo influenza A virus infection, A EC2 were isolated from C 57 BL six mice that were administered either phosphate buffered saline or influenza A virus PR eight A 34 intranasally one, two or three days earlier.These plots show lung cell suspensions from an uninfected as well as a three day infected C 57 black six males stained for sorting due to the recruitment of immune cells to the infected lung. The proportion of fluorescence negative side with scatter area high cells is substantially decreased within the cell suspension prepared from the three day infected compared to the uninfected mice. Influenza, A virus nuclear protein expression sorted a C two was analyzed through intracellular staining with a nuclear protein specific antibody.The histogram shows that a C two isolated from in vivo infected mice yield a specific fluorescence signal after intracellular staining for the influenza A virus, nucleo protein. The table shows the mean fluorescence intensity of the nuclear protein staining the intensity of the signal produced by the intracellular nuclear protein staining of a EC2 isolated from influenza. A virus infected mice increases with the duration of the viral infection to demonstrate the viability of a EC2 isolated from healthy mice and to test their susceptibility to influenza A virus infection.Influenza, a virus nuclear protein expression was also analyzed in isolated cells infected X vivo as shown here. A substantial proportion of a EC2 expresses the viral nuclear protein six hours post infection. These data are summarized here.The percentage of a EC2 positive for the intracellular staining of the viral nuclear protein depends on the viral dose used for the ex vivo infection experiment. These results show that the isolated primary neuron, a EC2, are viable and susceptible for viral infection and replication assessed as expression of viral protein seen. While attempting this procedure, it's very important to remember to carefully perform all tissue digestion and disintegration steps.Also, it's very important to remember to gait stringently on the fluorescence negative and side s getter area high cells. When sorting Following this procedure, other methods like molecular and functional studies can be formed to rule out the role of ular type two tthe third of the lung in the context of viral infections and autoimmune mediad lung diseases.I.

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Research

JoVE Journal

Immunology and Infection

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

Isolation of Mouse Lung Dendritic Cells

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Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

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Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

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Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

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Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

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Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

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Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

Isolation of Primary Mouse Lung Endothelial Cells

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Isolation of Live Myeloid and Epithelial Cell Populations from the Mouse Lung

This protocol presents a flow cytometry-based method to isolate viable alveolar type II epithelial cells (AECII) from neonatal, juvenile, or adult murine lungs (infected or non-infected). This approach enables downstream molecular and/or functional studies of AECII to identify their roles during lung inflammation and infection.

Isolation of Alveolar Type II Epithelial Cells from Neonatal, Juvenile, and Adult Murine Lungs Adaptable to Infectious Experimental Settings

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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