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

Podsumowanie

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.

Streszczenie

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 (SSC^high) 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}.

Protokół

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 °C in a water bath. In a heating block, shortly heat small aliquots of 1 % low-melt agarose (in water) to 95 °C until liquefied and subsequently cool to 45 °C until use.

1. Preparation of the Mouse Lung

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. Cut and remove the salivary glands to expose the trachea. Also carefully cut the muscle surrounding the trachea.
7. 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.

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.

1. 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.
2. 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.
3. 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.
4. Put the lung into the remaining 2 ml of dispase and incubate at room temperature for 45 min.

3. Preparation of the Lung Cell Suspension

1. Remove the lung from the dispase and transfer it into a dish containing 7 ml of anti-CD16/32 antibody (1 μg/ml final concentration) in DMEM medium and 100 μl DNase.
2. 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 °C until the next step.
3. Filter the cell suspension through nylon meshes first with 100 μm and subsequently with 70 μm, 48 μm and 30 μm 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.
4. 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 °C. Remove the supernatant.
5. 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 °C.

4. Antibody Staining for Flow Cytometric Cell-sorting

1. 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 μl. 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).
2. For staining, incubate the cells for 10 min in the dark at 4 °C.
3. Wash the cells by filling the 15 ml tube with DMEM medium and centrifugation for 12 min at 160 x g and 4 °C.
4. 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 °C in the dark.
5. Wash the cells through filling the tube with DMEM and centrifugation for 12 min at 160 x g and 4 °C.
6. Resuspend the cells in 1 ml of DMEM and pre-filter through a 50 μm filter into a tube for cell-sorting. Optional: Count the cells to obtain the total cell number in the lung cell suspension.

5. Cell-sorting

1. Mix the cells by vortexing prior to sorting. Use a 100 μm 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.
2. Gate on the SSC^high 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).
3. In order to collect the sorted cells centrifuge for 20 min at 280 x g and 4 °C.
4. Resuspend the cells in culture-medium or buffer as required for subsequent analysis.

Wyniki

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 i...

Dyskusje

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 ...

Materiały

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