Name: Isolation of Alveolar Type II Epithelial Cells from Neonatal, Juvenile, and Adult Murine Lungs Adaptable to Infectious Experimental Settings (Video) | JoVE Journal
Uploaded: 2025-12-19T14:10:00.000+00:00
Duration: 6 min 14 s
Description: Learn a flow cytometry-based method to isolate alveolar type II epithelial cells from murine lungs at different developmental stages. This protocol enables infection studies and supports research on lung inflammation.

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5 minutes

The authors would like to thank Tatjana Hirsch and Friederike Kruse for their expert technical assistance, Lisa H&#246;nicke and Justine Smout for their helpful discussions, and Yoshihiro Kawaoka for providing the IAV-mCherry reporter virus. This study was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - TRR 359 - Project number 491676693 (to D.B. and J.H.), the COVIPA initiative (KA1-Co-02, Helmholtz Association; to D.B.), and a grant from the German Research Foundation (BO 7036/1-1; to J.D.B.). We acknowledge support from the Open Access Publication Fund of the Medical Faculty of the Otto-von-Guericke-University Magdeburg.

All animals were bred and housed in the animal facility at the Helmholtz Centre for Infection Research under specific-pathogen-free (SPF) conditions and in accordance with national and institutional guidelines. Euthanasia of na&#239;ve mice for subsequent AECII isolation was performed in accordance with national guidelines from the Society of Laboratory Animal Science (GV-SOLAS, Gesellschaft f&#252;r Versuchstierkunde). Male mice on a C57BL/6 genetic background at an age of 2 days (neonatal), 3 weeks (juvenile), or 8-12 weeks (adult) were used. The reagents and the equipment used are listed in the Table of Materials.
1. Preparation of the mouse lung
<ol>
	<li>Sacrifice the mouse (following institutionally approved protocols):
	<ol>
		<li>For adult and juvenile mice: administer an anesthesia overdose (e.g., ketamine/xylazine) via intraperitoneal injection.</li>
		<li>For neonatal mice: decapitate. 
		NOTE: Avoid cervical dislocation in adult and juvenile mice as this procedure can damage the trachea.</li>
	</ol>
	</li>
	<li>Sterilize the mouse fur with 70% (v/v) ethanol. 
	CAUTION: Inflammable liquid. Eye irritant. Keep away from heat. Keep the container tightly closed, and make a ventral midline incision to expose internal organs.</li>
	<li>Carefully remove the skin and peritoneum.</li>
	<li>Exsanguinate the mouse by cutting both jugular veins and the vena cava, allowing blood to drain from the body.</li>
	<li>Carefully remove the diaphragm to expose the heart and lungs. 
	NOTE: Take care not to injure the lungs. Perforation of lung tissue would significantly impede the efficiency of subsequent enzyme-mediated digestion of lung tissue.</li>
	<li>Open the ribcage using scissors.</li>
	<li>Insert a cannula into the right ventricle of the heart and perfuse with cold phosphate-buffered saline (PBS) until residual blood is cleared from the lung and lung tissue becomes pale:
	<ol>
		<li>For adult mice: use a 26 G cannula and perfuse the lung with 10 mL PBS.</li>
		<li>For neonatal mice: use a 30 G cannula and perfuse the lung with 1 mL PBS. 
		NOTE: Do not perfuse the lungs of juvenile mice to avoid rupture of the alveolar structure by excessive pressure in pulmonary blood vessels. Perfusion might be less effective in inflamed lungs, and the change of color in lung tissue might be less pronounced. If required, a bronchoalveolar lavage can be performed prior to the following step in adult and juvenile mice.</li>
	</ol>
	</li>
</ol>
2. Enzymatic digestion of lung tissue
<ol>
	<li>Adult and juvenile mice:
	<ol>
		<li>Expose the trachea by removing the salivary glands and the surrounding muscle.</li>
		<li>Insert a 22 G indwelling cannula into the trachea, remove its needle, and move the plastic catheter towards the lungs.</li>
		<li>Securely tie a piece of yarn around both trachea and catheter to hold it in place.</li>
		<li>Prepare the lung tissue by instilling dispase. 
		CAUTION: May cause allergy or asthma symptoms or breathing difficulties if inhaled. Avoid breathing dust. Wear respiratory protection. Dispose of contents/ container to an approved waste disposal plant.) through the catheter inserted into the trachea:
		<ol>
			<li>For adult mice: Insert 2 mL of dispase.</li>
			<li>For juvenile mice: Insert 500 &#181;L of dispase. 
			NOTE: Use a syringe with a luer slip connection to facilitate exchange of syringes between steps 2.1.4 and 2.1.5. To ensure efficient enzymatic digestion of the lung tissue, it is vital to ensure complete enzyme filling of bronchoalveolar spaces.</li>
		</ol>
		</li>
		<li>Exchange the syringe and repeat with low-melt agarose (1% (w/v), 45 &#176;C) to maintain structure during processing.
		<ol>
			<li>For adult mice: Use 500 &#181;L of low-melt agarose.</li>
			<li>For juvenile mice: Use 200 &#181;L of low-melt agarose. 
			NOTE: To avoid backflow of the fluids, leave the syringe attached. 
			NOTE: Before use: dissolve low-melt agarose using heat (95 &#176;C), let agarose solution cool down to 45 &#176;C, e.g., in a thermoblock.</li>
		</ol>
		</li>
		<li>Cover the lung with laboratory tissue paper and add ice to allow the agarose to solidify for 2 min.</li>
		<li>Remove ice, tissue paper, syringe, and catheter.</li>
		<li>Tie the yarn to close the trachea in order to keep the enzyme in the lower airways.</li>
		<li>Remove heart and thymus.</li>
		<li>Cut the trachea above the yarn and remove the intact lung from the chest.</li>
		<li>Transfer the lung into a 15 mL tube containing pre-warmed dispase (37 &#176;C) and incubate the lung tissue in dispase for 45 min at room temperature (20-25 &#176;C).
		<ol>
			<li>For adult mice: incubate the lung in 2 mL dispase.</li>
			<li>For juvenile mice: incubate the lung in 1.5 mL dispase.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Neonatal mice
	<ol>
		<li>Collect and mince single lung lobes using fine scissors in the lid of a 2 mL tube.</li>
		<li>Add 500 &#181;L dispase, close the lid, and invert the tube to ensure that the entire tissue is covered in dispase.</li>
		<li>Incubate the lung tissue in dispase at room temperature for 45 min.</li>
	</ol>
	</li>
</ol>
3. Preparation of the lung tissue
<ol>
	<li>For adult and juvenile mice:
	<ol>
		<li>Remove the lung from the dispase and dissect the lung lobes from the bronchial stem.</li>
		<li>Place the lung lobes into a Petri dish (diameter: 8.5 cm) containing 7 mL DMEM + HEPES, 1 &#181;g/mL (final concentration) anti-CD16/32 blocking antibody and 100 &#181;L DNAse (final concentration ~600 Kunitz units). 
		CAUTION: May cause allergy or asthma symptoms or breathing difficulties if inhaled. Avoid breathing dust. Wear respiratory protection. Dispose of contents/container to an approved waste disposal plant.</li>
		<li>Disintegrate the lung tissue using forceps. 
		NOTE: To ensure efficient enzymatic digestion of the lung tissue, it is vital to ensure thorough mechanical tissue disintegration.</li>
		<li>Incubate for 10 min at room temperature while gently rocking at 200 rpm. 
		NOTE: Cell suspension can be stored at 4 &#176;C until the next step, if required.</li>
	</ol>
	</li>
	<li>For neonatal mice:
	<ol>
		<li>Transfer the dispase, including the digested tissue, to a Petri dish (diameter: 5.4 cm) containing 3 mL DMEM + HEPES, 1 &#181;g/mL (final concentration) anti-CD16/32 blocking antibody and 100 &#181;L DNAse (final concentration ~ 600 Kunitz units).</li>
		<li>Incubate for 10 min at room temperature while gently rocking at 200 rpm. 
		NOTE: Cell suspension can be stored at 4 &#176;C until the next step, if required.</li>
	</ol>
	</li>
	<li>Continue for all age groups:
	<ol>
		<li>Filter the cell suspension through a series of nylon cell strainers with decreasing pore size (100 &#181;m - 70 &#181;m - 40 &#181;m) to remove debris and large aggregates.</li>
		<li>Carefully rinse cell strainers and tubes to minimize loss of cells. In case samples are pooled, cell suspensions of mice from the same group can be passed through the same cell strainers. 
		NOTE: In case of clogging, change the cell strainers.</li>
		<li>Depending on the volume, transfer the filtered cell suspension to one or more 15 mL tubes and centrifuge the cell suspension for 12 min at 140 x g at 4 &#176;C.</li>
		<li>Discard the supernatant and resuspend the cell pellet in erythrocyte lysis buffer (ACK buffer).
		<ol>
			<li>For adult and juvenile mice: Use 2 mL of erythrocyte lysis buffer and incubate for 2 min.</li>
			<li>For neonatal mice: Use 3 mL of erythrocyte lysis buffer and incubate for 3 min.</li>
		</ol>
		</li>
		<li>Stop erythrocyte lysis by filling the 15 mL tube with DMEM + HEPES + 10% (v/v) FBS and centrifuge again for 12 min at 140 x g at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
4. Antibody staining for fluorescence-activated cell sorting
<ol>
	<li>Resuspend the cells in an antibody staining solution containing anti-F4/80, anti-CD93, anti-CD11c, anti-CD19, anti-CD31, anti-CD11b, anti-CD16/32, anti-CD45, anti-CD24 and anti-CD326 fluorochrome-coupled antibodies prepared in DMEM + HEPES + 10% (v/v) FBS. 
	NOTE: For optimal staining results, use only pre-titrated antibodies. If AECII are required to be untouched for cell sorting, do not include anti-CD326 in the antibody staining solution.
	<ol>
		<li>For adult mice: Use 600 &#181;L antibody solution per lung.</li>
		<li>For juvenile mice: Use 400 &#181;L antibody solution per lung.</li>
		<li>For neonatal mice: Use 200 &#181;L antibody solution per lung.</li>
	</ol>
	</li>
	<li>Incubate the cell suspension with the antibodies for 10 min at 4 &#176;C in the dark.</li>
	<li>After incubation, wash the cells by filling the tube with DMEM + HEPES + 10% (v/v) FBS.</li>
	<li>Centrifuge for 12 min at 140 x g at 4 &#176;C.</li>
	<li>Resuspend the cells DMEM + HEPES + 10% (v/v) FBS:
	<ol>
		<li>For adult mice: Resuspend in 300 &#181;L per lung.</li>
		<li>For juvenile mice: Resuspend in 200 &#181;L per lung.</li>
		<li>For neonatal mice: Resuspend in 100 &#181;L per lung.</li>
	</ol>
	</li>
</ol>
5. Sorting of AECII
NOTE: Instrument preparation: install a 100 &#181;m nozzle for sorting AECII using a FACS device.
<ol>
	<li>Directly prior to sorting, filter cells through a 50 &#181;m cell strainer. Rinse the cell strainer with DMEM supplemented with HEPES and 10% (v/v) FBS to prevent excessive cell loss:
	<ol>
		<li>Adult mice: Rinse the cell strainer with 300 &#181;L per lung.</li>
		<li>Juvenile mice: Rinse the cell strainer with 200 &#181;L per lung.</li>
		<li>Neonatal mice: Rinse the cell strainer with 100 &#181;L per lung.</li>
	</ol>
	</li>
	<li>Resuspend the cells by vortexing directly before sorting.</li>
	<li>Gate for AECII by identifying events that I) have high side scatter (SSChigh), II) are negative for the lineage fluorochromes, and III) are positive for the CD326 fluorochrome coupled to the anti-CD326 antibody. 
	NOTE: Use fluorochromes with high-to-very-high brightness indices (such as APC, PE, and BV785) to facilitate discrimination between non-AECII and AECII. For an untouched sorting approach (exclusion of anti-CD326 in the staining solution), AECII are identified as lineage-negative, SSChigh, and autofluorescencepositive/high cells. AECII autofluorescence can be identified in green light detectors, e.g., FITC or GFP detectors.</li>
	<li>Exclude doublets or aggregates using forward scatter height vs. area (FSC-H vs. FSC-A) and sideward scatter height vs. area (SSC-H vs. SSC-A).</li>
	<li>Collect the sorted cells into a tube containing DMEM supplemented with HEPES and 10% (v/v) FBS and centrifuge for further analysis or culture.</li>
</ol>
6. RNA-Isolation from PFA-fixed AECII (Optional)
NOTE: In case AECII shall be isolated from mice that had been infected with, e.g., respiratory viral pathogens, AECII isolation needs to be carried out under BSL2 or BSL3 conditions, depending on the pathogen. If the experimenter does not have access to cell sorting under the appropriate safety conditions, AECII isolated using the protocol described above can be fixed with PFA after antibody staining. This step inactivates pathogens10,11 and thus subsequent flow cytometric AECII sorting can take place under BSL1 conditions. Although the cells can no longer be employed for functional assays due to the fixation process, they are suitable for molecular analyses.
<ol>
	<li>For inactivation of viruses, fix antibody-labeled single cell suspensions in 500 &#181;L of 4% (w/v) PFA. 
	CAUTION: Flammable solid. Harmful if swallowed or inhaled. Causes skin irritation, serious eye damage. May cause an allergic skin reaction, respiratory irritation, and cancer. Suspected of causing genetic defects. Keep away from heat. IF SWALLOWED: Call a POISON CENTER/ doctor if you feel unwell. IF INHALED: Remove person to fresh air and keep them comfortable for breathing. Call a POISON CENTER/ doctor if you feel unwell. IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. IF EXPOSED or CONCERNED: Get medical advice/ attention. 
	NOTE: In compliance with the 3R guidelines, AECII from non-infected mice were used in this study. AECII were fixed for 10 min at room temperature. The duration of fixation must be tested individually for each pathogen that poses a potential risk to humans and the environment. Subsequent sorting under S1 conditions requires prior verification in a BSL2 or BSL3 laboratory that no replicable virus is present after PFA fixation.</li>
	<li>After fixation, wash cells with DMEM and centrifuge for 15 min at 140 x g.</li>
	<li>Resuspend the cells in 300 &#181;L DMEM + HEPES + 10% (v/v) FBS and pass the cell suspension through a 100 &#181;m cell strainer.</li>
	<li>Rinse the cell strainer with an additional 300 &#181;L DMEM + HEPES + 10% (v/v) FBS medium to prevent excessive cell loss.</li>
	<li>Sort cells as described in step 5. &#34;Sorting of AECII&#34;. 
	NOTE: After mandatory experimental verification that any virus was inactivated by treatment with 4% (w/v) PFA, AECII can be sorted under BSL1 conditions.</li>
	<li>For RNA isolation from PFA-fixed cells, use the RNeasy FFPE Kit. 
	CAUTION: This kit contains hazardous chemicals. Follow the instructions by the manufacturer. Pay attention to the included safety data sheet, with the following adjustments:
	<ol>
		<li>After sorting, centrifuge the cells for 15 min at 140 x g at 4 &#176;C.</li>
		<li>Resuspend the cells in 150 &#181;L Buffer PKD and transfer into a 1.5 mL tube.</li>
		<li>Add 10 &#181;L of Proteinase K and incubate at 56 &#176;C for 15 min, followed by incubation at 80 &#176;C for up to 15 min, depending on the cell count (see Table 1).</li>
	</ol>
	</li>
	<li>Continue with the RNA isolation process as per the protocol, including DNase treatment, washing steps, and final elution in 30 &#181;L RNase-free water.</li>
</ol>

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<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+%22Respiratory+Res%22%5BJournal%5D)">Alveolar epithelial type II cell: Defender of the alveolus revisited.</a> Respiratory Res. 2 (1), 33(2001).</li>
<li>Mason, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biology+of+alveolar+type+II+cells%5BTitle%5D)+AND+%22Respirology%22%5BJournal%5D)">Biology of alveolar type II cells.</a> Respirology. 11 (Suppl), S12-S15 (2006).</li>
</ol>

The authors declare no conflicts of interest.

The optimized protocol outlined here provides a reliable and reproducible approach for the isolation and analysis of AECII from mouse lung tissue at neonatal, juvenile, and adult ages. Furthermore, it provides an optional PFA fixation step prior to sorting, which allows AECII to be sorted from mice under BSL1 conditions, even if they have previously been infected with respiratory pathogens. Through the combination of carefully carried out lung exposure and perfusion, followed by intratracheal instillation of enzymes and low-melting agarose into the lung, tissue structure is maintained, enabling recovery of high-quality single-cell suspensions for downstream analysis. The addition of flow cytometric sorting on side-scatter properties, lineage-negative gating, and positive selection for CD326-positive cells resulted in remarkably pure populations of AECII. Such purity is of particular importance in ensuring that molecular and functional assays downstream are representative of intrinsic AECII biology and not confounded by contaminating cell types, enhancing the interpretability of gene-expression and virus-host interaction studies.
The abovementioned protocol features exclusion of non-AECII using a wide array of fluorescently-labelled antibodies directed against leukocytic and non-leukocytic antigens and subsequent positive selection of AECII via side-scatter properties and expression of the epithelial-cell specific molecule CD326. While it was shown that this methodology resulted in highly pure AECII populations (using SPC, a hallmark AECII marker), the purity of the obtained populations could only be confirmed using post-sort intracellular staining, thus far. This protocol can be applied to isolate AECII from SARS-CoV-2 and Influenza virus-infected lungs, in which AECII are primarily targeted by the virus and display distinct cellular adaptations1. However, this method may not be appropriate to isolate murine AECII in the context of lung cancer biology studies, as EpCAM (CD326) is also expressed by pulmonary cancer cells in mice and humans13. For these studies, the use of transgenic mouse strains that express fluorescent proteins under the transcriptional control of the Spc promoter may provide a more suitable approach for the reliable isolation of primary AECII.
A yield of 0.15 &#215; 106 viable AECII per neonatal lung and up to 1.0 &#215; 106 cells per adult lung illustrates the efficiency and scalability of the protocol. A major obstacle to the successful isolation of large AECII quantities is inefficient enzymatic digestion and mechanical disintegration of the lung tissue. Thus, it is crucial to ensure complete filling of bronchoalveolar spaces with the enzyme in step 2.1.4 and to carefully perform mechanical tissue disintegration in step 3.1.3. Effective enzymatic digestion yields a turbid cell suspension with minimal contribution of visible aggregates that usually can be disintegrated using a plunger in the following filter steps. Larger aggregates that cannot be passed through the cell strainer usually arise as a consequence of insufficient enzymatic digestion. Note that it is not advised to prolong the enzymatic digestion times, as this may negatively affect subsequent immunostaining14 and thus the accuracy of FACS-based AECII isolation.
Next to the efficiency of enzymatic and mechanical tissue disintegration, the genetic background may impact AECII yield. For this protocol, mice on a C57BL/6 background were used. In line with reported developmental15, immunological16, and physiological17,18,19 strain-specific differences, it is conceivable that the use of other laboratory mouse strains (e.g., BALB/c, DBA) may result in different AECII yields. Moreover, AECII yield from inflamed lungs (infectious or non-infectious) can be significantly reduced. This can be the result of indirect (inflammatory) or direct (pathogen-mediated) cell death of AECII that represent major targets for e.g., IAV20,21 and SARS-CoV-222,23,24.
The preservation of cellular integrity during mechanical dissociation, enzymatic digestion, and flow sorting is further key for downstream molecular and/ or functional analyses. Therefore, AECII viability and cellular function were assessed post-sorting. AECII were capable of adhering to Matrigel and maintaining a characteristic lamellar body-rich morphology for at least 24 h, which indicates functional competence following isolation and suitability for ex vivo culture (including infection models).
The efficient infection of sorted AECII with IAV-mCherry reporter virus12 and the observation of strong viral replication validate this system as a robust platform for elucidating epithelial-intrinsic antiviral responses. Since AECII regulate key processes involved in surfactant biosynthesis, alveolar regeneration, and innate immunity25,26, the capacity to examine IAV replication kinetics and host transcriptional responses in purified AECII populations will provide insights not obtainable in mixed-cell preparations.
A key innovation of this protocol is the inclusion of a fixation procedure, allowing side-by-side analysis of fresh and inactivated samples. The PFA fixation and adapted FFPE RNA extraction processes maintain RNA integrity to a degree compatible with downstream transcriptomic analyses, as evidenced by successful quantitative reverse-transcription PCR (qPCR).
PFA fixation times may affect the quality of staining and thus the recovery of AECII by FACS. Moreover, the yield and quality of RNA are generally affected by fixation processes. Depending on the in vivo infection model utilized, these aspects need to be considered for experimental design. It is recommended to avoid extensive fixation time and instead optimize the duration of PFA treatment so that the minimum necessary time is used, which has been experimentally proven to ensure complete inactivation of the respective virus.
Assessment of the abundance of ribosomal RNA (rRNA) using a microfluidics instrument serves as a surrogate to estimate mRNA quality in biological samples. The outlined AECII fixation/RNA isolation protocol allows the isolation of intact RNA with sufficient quality for downstream transcriptomic analyses, as evidenced by 28S/18S rRNA ratios &#62;1 and RNA quality number (RQN) values &#62;8. It is important to note that the 80 &#176;C incubation step critically reduces RNA yield and quality. Adaptation of thermal treatment times can optionally be applied to increase RNA yield and quality for downstream applications.
Collectively, these methodological improvements provide an extensive toolkit for the study of AECII biology in the context of health, infectious disease, and development (newborn to adult). The high yield and purity of AECII isolated by this approach allow for multi-omic profiling, encompassing transcriptomic, proteomic, and chromatin accessibility assays, as well as functional assays (e.g., ex vivo infection) to further dissect relevant mechanisms of AECII in vivo.

Due to their multifaceted roles in lung development, physiology, immunity, and repair processes, alveolar type II epithelial cells (AECII) vitally contribute to the maintenance as well as to the re-establishment of homeostasis following lung injury. As AECII are primary cellular targets of pandemic viruses such as influenza A virus (IAV) or SARS-CoV-2, their fate and functions have been in the spotlight of infection biology research. In this context, previous studies reported that intrinsic (e.g., genetic status1, age2,3) as well as extrinsic (e.g., microbial stimuli4) cues have a major impact on AECII phenotypes and their responsiveness to environmental triggers such as airborne pathogens. Notably, pulmonary inflammation has been shown to leave sustained changes in transcriptomic, proteomic, and epigenetic status of AECII5,6. The complex cellular interactions of AECII within the lung, as well as the contribution of various biotic and abiotic factors to AECII biology, highlight the importance of suitable in vivo models that allow for examining these cells in more detail. 
FACS represents the current gold standard technique for the enrichment and purification of primary cells from human and animal tissues, as it enables the simultaneous implementation of multiparametric selection (or exclusion) and represents the only method, in which cell-intrinsic properties like size, granularity, or autofluorescence can be reliably integrated in the selection/exclusion workflow. To date, a few published protocols provide methodologies for FACS-based isolation of primary murine AECII. Sinha and Lowell reported a method for AECII isolation from adult mice that features similar tissue processing and flow cytometry workflow and included enrichment of AECII prior to FACS by bead-based magnetic depletion on non-target cells7. Another protocol describes the use of magnetic-bead-based isolation of EpCAM-positive lung cells or the use of transgenic mice that allowed for FACS-based isolation of AECII based on identification of SPC-positive (GFP+) cells using a conditional reporter mouse model8. While SPC expression reliably identifies AECII in murine lungs, this methodology is only applicable in combination with a dedicated mouse reporter strain and thus precludes AECII isolation from, e.g., wild-type mice. Importantly, while similar yields and purity of AECII were reported, none of these protocols provided adaptations for the isolation of AECII from neonatal, juvenile, or infected hosts. 
To this end, the present protocol includes a set of adapted workflows for the isolation of highly pure and viable AECII from mice using enzymatic digestion in combination with FACS. This state-of-the-art methodology is based on a previously developed FACS-based AECII isolation protocol9 and contains important refinements that improve AECII purity through the addition of further identification and exclusion markers. Importantly, this updated protocol features AECII retrieval via both "touched" and "untouched" sorting strategies. Moreover, as postnatal lung development is associated with major changes regarding cellular composition and structural reorganization, this protocol includes adaptations to optimize AECII retrieval from neonatal, juvenile, and adult lungs. This allows one to examine AECII biology during ontogeny, e.g., in the context of lung maturation or infection biology studies. This approach can be used to obtain viable AECII for subsequent ex vivo cultivation and infection with IAV. Additionally, this protocol includes a fixation procedure using the chemical fixative paraformaldehyde (PFA). This step warrants the inactivation of various respiratory pathogens (e.g., SARS-CoV-2, IAV) in lung cell suspensions, which in turn allows subsequent sample handling (including FACS-based AECII isolation) in facilities with lower biocontainment precautions. While the PFA-based fixation precludes subsequent AECII ex vivo cultivation for functional studies, it allows the retrieval of significant amounts of intact RNA for transcriptomic profiling of AECII to elucidate their biological roles in the context of respiratory infections.

<table><tbody><tr><td>ACK buffer</td><td>-</td><td>-</td><td>8.29 g NH&lt;sub&gt;4&lt;/sub&gt;Cl, 1 g KHCO&lt;sub&gt;3&lt;/sub&gt;, 200 &amp;micro;L of 0. 5 M EDTA, add 1000 mL Milli-Q, adjust pH to 7.2 to 7.4</td></tr><tr><td>anti-mouse CD11b</td><td>eBioscience</td><td>12-0112-83</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 CD16/32</td><td>BD Biosciences</td><td>553141</td><td>clone 93; purified</td></tr><tr><td>anti-mouse CD16/32</td><td>BD Biosciences</td><td>561727</td><td>clone 2.4G2; PE coupled</td></tr><tr><td>anti-mouse CD19</td><td>Biolegend</td><td>115508</td><td>clone 6D5; PE coupled</td></tr><tr><td>anti-mouse CD24</td><td>Biolegend</td><td>101808</td><td>clone M1/69; PE coupled</td></tr><tr><td>anti-mouse CD31</td><td>Biolegend</td><td>102408</td><td>clone 390; PE coupled</td></tr><tr><td>anti-mouse CD326</td><td>Biolegend</td><td>118245</td><td>clone G8.8; BV785 coupled</td></tr><tr><td>anti-mouse CD45</td><td>BD Biosciences</td><td>553081</td><td>clone 30-F11; PE coupled</td></tr><tr><td>anti-mouse CD93</td><td>eBioscience</td><td>17-5892-82</td><td>clone AA4-1; APC coupled</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 SPC&amp;nbsp;</td><td>abcam</td><td>ab211326</td><td>host species: rabbit; clone: EPR19839; purified</td></tr><tr><td>Aria-Fusion</td><td>BD Biosciences</td><td></td><td>Cell Sorter</td></tr><tr><td>Aria-II SORP</td><td>BD Biosciences</td><td></td><td>Cell Sorter</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>Cell strainer (100 &amp;micro;m, 70 &amp;micro;m, 40 &amp;micro;m)</td><td>BD Falcon</td><td>352360, 352350, 352340</td><td></td></tr><tr><td>CellTrics (50 &amp;micro;m)</td><td>Sysmex</td><td>04-004-2327</td><td></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 330 &amp;micro;L DMEM</td></tr><tr><td>Dispase, 100 mL (5000 caseinolytic units)</td><td>Corning</td><td>354235</td><td>perform sterile filtration (pore size 0.22 &amp;micro;m), prepare 4 mL aliquots in 15 mL tubes, store at -20 &amp;deg;C</td></tr><tr><td>DMEM</td><td>Gibco</td><td>31885-049</td><td>low glucose, with pyruvate, add HEPES (final concentration of 25 mM)</td></tr><tr><td>FACS Diva</td><td>BD Biosciences</td><td></td><td>Sorting Software</td></tr><tr><td>FlowJo</td><td></td><td></td><td>Evaluation Software for FACS data</td></tr><tr><td>goat anti-rabbit</td><td></td><td></td><td>Alexa 594 coupled</td></tr><tr><td>Indwelling cannula Introcan 22G</td><td>Braun</td><td>4252098B</td><td></td></tr><tr><td>RNeasy FFPE Kit</td><td>Qiagen</td><td>73504</td><td></td></tr><tr><td>Symphony S6 SE</td><td>BD Biosciences</td><td></td><td>Cell Sorter</td></tr></tbody></table>

isolation of alveolar type ii epithelial cells from neonatal, juvenile, and adult murine lungs adaptable to infectious experimental settings

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

Alveolar type II epithelial cells (AECII) critically contribute to immune regulation in the lung and thus are key elements orchestrating pulmonary homeostasis. These functions render AECII particularly relevant in the context of infection with respiratory viruses, which target the lower airway epithelium and disrupt alveolar integrity. Preclinical mouse models are vital tools to elucidate the roles of AECII in the context of lung inflammation and/or infection. This protocol presents a detailed and reproducible pipeline for isolating viable and highly purified AECII from murine lungs at neonatal, juvenile, and adult stages. The methodology combines mechanical dissociation, enzymatic digestion, and subsequent fluorescence-activated cell sorting (FACS) to yield highly enriched AECII populations proven to be suitable for downstream molecular and functional analyses. For applications involving pathogenic viruses, the protocol includes an optional paraformaldehyde (PFA) fixation step, enabling safe handling under lower biosafety conditions without compromising RNA integrity. With optimized cell recovery across developmental stages, yielding up to 1.0 &#215; 106 AECII cells per adult lung, this protocol supports preclinical studies of, e.g., viral pathogenesis, epithelial function, and regenerative capacity.

The protocol outlined here represents an optimized and detailed method for isolating and analyzing AECII from mouse lung tissue at various developmental stages, ranging from neonatal to juvenile and adult mice. The methodology involves mechanical and enzymatic lung dissociation, combined with an optimized flow cytometric cell sorting workflow, utilizing custom protocols suitable for processing samples from infected animals for subsequent Flow Sorting under BSL1 conditions.
The experimental data revealed a high efficacy of this isolation procedure (summarized in Figure 1) in the production of viable and highly pure (&gt;95%) AECII populations. Flow-sorting generated pure AECII populations, with post-sort analysis validating almost 100% specificity for established cellular markers (Figure 2). Quantification of post-sort cell samples indicated high yields between 0.15 × 106 viable AECII from neonatal lungs and 1.0 × 106 cells from adult samples per whole lung preparation (Figure 3A). Isolated AECII maintained typical morphology (Figure 3B) and cellular integrity throughout the isolation process (Figure 3C). Additionally, the applicability of the protocol for functional studies was confirmed through successful ex vivo infection using a mouse-adapted IAV (H1N1, strain A/Puerto Rico/8/34) expressing the mCherry reporter protein (IAV-mCherry12), with a demonstration of the ability of the cells to support viral replication (Figure 4).
To support experimental workflows that necessitate sample fixation, the protocol includes a specialized RNA extraction approach using PFA fixation. Thorough examination of RNA quality from PFA-fixed AECII at different processing time points indicated that high-quality RNA amenable to downstream molecular analyses can be reliably extracted from fixed samples (Table 1). The quality of extracted RNA showed a direct relationship with the duration of thermal treatment at 80 °C during the extraction procedure, offering clear optimization guidelines based on particular experimental needs (Table 1, Figure 5). Validation by quantitative reverse transcription PCR (qRT-PCR) analysis showed successful amplification of both housekeeping genes (ribosomal protein S9, Rps9) and AECII-specific markers (surfactant protein C, Sftpc) with PFA-fixed samples and fresh control preparations (Figure 6).
<img alt="Mouse lung preparation flowchart; neonatal, juvenile, adult tissue digestion, FACS analysis, staining." class="xfigimg" src="/files/ftp_upload/69581/69581fig1.jpg" data-altupdatedat="1778558422000" data-alt="Figure 1"> 
Figure 1: Schematic workflow for AECII isolation from neonatal, juvenile and adult mouse lungs using enzymatic tissue digestion and flow cytometric cell sorting. The diagram illustrates the systematic processing of mouse lungs from different age groups. Euthanasia of mice and lung extraction is followed by multi-step enzymatic digestion with dispase for tissue disintegration. Following mechanical disruption, cells are purified through a series of filtration steps with decreasing pore sizes. Subsequently, erythrocytes are lysed. Specific enrichment of AECII is achieved through negative selection using antibody labeling against hematopoietic markers (CD45, CD11b, CD11c, F4/80, CD19, CD16/32), endothelial marker CD31, platelet marker CD93 and bronchial epithelial cell marker CD24, positive selection of epithelial cells using an antibody labeling against CD326 and subsequent flow cytometric sorting based on the characteristic SSChigh profile of granular AECII. This protocol enables the isolation of highly pure (≥ 95%), viable primary AECII for downstream functional and molecular analyses. <a href="https://www.jove.com/files/ftp_upload/69581/69581fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Flow cytometry and microscopy analysis of lung cells; pre/post-sort scatter plots and dye staining." class="xfigimg" src="/files/ftp_upload/69581/69581fig2.jpg" data-altupdatedat="1778558422000" data-alt="Figure 2"> 
Figure 2: Representative gating strategy used for the isolation of AECII from adult mice. (A) Lung single cell suspensions were stained with antibodies against the following antigens: F4/80, CD93, CD11c (coupled to APC), and CD19, CD31, CD11b, CD16/32, CD45, CD24 (coupled to PE), and CD326 (coupled to BV785). (B) Re-analysis (A) for purity analysis. Sorted AECII were re-analyzed in order to confirm 97.2% purity of AECII. (C) Representative fluorescence microscopy images of AECII after sorting. Nuclei were stained with DAPI (blue). Intracellular staining was performed using a primary anti-SPC antibody (species: rabbit), and secondary staining was performed using goat anti-rabbit IgG coupled to Alexa 594 (red). AECII stained with DAPI and secondary antibody only, and splenocytes stained with primary and secondary antibodies served as relevant controls, indicating specific binding of anti-SPC as well as anti-rabbitAlexa 594. Scale bars: 20 µm. <a href="https://www.jove.com/files/ftp_upload/69581/69581fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bar graph showing lung cell viability; microscope images of AECII in neonatal, juvenile, adult samples." class="xfigimg" src="/files/ftp_upload/69581/69581fig3.jpg" data-altupdatedat="1778558422000" data-alt="Figure 3"> 
Figure 3: Yield, morphology and vitality of AECII after fluorescence-activated cell sorting. (A) Total number of viable AECII per lung obtained from neonatal, juvenile, and adult mice. (B) Light-microscopy image of sorted AECII post sorting. Scale bar: 75 µm. (C) AECII viability observed by trypan blue staining after sorting. <a href="https://www.jove.com/files/ftp_upload/69581/69581fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fluorescence microscopy, IAV-mCherry infection results, control vs infected, timepoints 0h and 8h." class="xfigimg" src="/files/ftp_upload/69581/69581fig4.jpg" data-altupdatedat="1778558422000" data-alt="Figure 4"> 
Figure 4: Ex vivo infection of sorted AECII with IAV-mCherry. AECII cells isolated from adult mice were cultivated ex vivo in Matrigel-coated tissue culture plates overnight to allow attachment. Subsequently, the cells were infected with IAV-mCherry (multiplicity of infection 2) for 8 h. A time-dependent increase in mCherry signal intensity was observed. Scale bar: 200 µm. <a href="https://www.jove.com/files/ftp_upload/69581/69581fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Heat-reacted PFA fixed AECII: Protein analysis graph. Charts compare control, 5-15 min at 80°C." class="xfigimg" src="/files/ftp_upload/69581/69581fig5.jpg" data-altupdatedat="1778558422000" data-alt="Figure 5"> 
Figure 5: RNA integrity from 4% PFA fixed AECII. Representative RNA profiles of PFA fixed and sorted AECII after RNA isolation using the Qiagen FFPE RNeasy Kit with modifications. Specific peaks for 18S and 28S rRNA, together with the RNA quality number (RQN), represent the RNA integrity and quality for intact isolated RNA from PFA fixed AECII. <a href="https://www.jove.com/files/ftp_upload/69581/69581fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="qPCR amplification curves; fluorescence vs. cycles; comparison of Rps9 and Sftpc; control vs. 4% PFA." class="xfigimg" src="/files/ftp_upload/69581/69581fig6.jpg" data-altupdatedat="1778558422000" data-alt="Figure 6"> 
Figure 6: Amplification curves of the Rps9 housekeeping gene and AECII-specific gene Sftpc from control (untreated) and 4% PFA fixed AECII. <a href="https://www.jove.com/files/ftp_upload/69581/69581fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample</td>
			<td>Incubation time at 80 °C</td>
			<td>28S/18S</td>
			<td>RQN</td>
			<td>RNA conc. [ng/µ]</td>
		</tr>
		<tr>
			<td>Control 0,5x106</td>
			<td>-</td>
			<td>2</td>
			<td>10</td>
			<td>5.8</td>
		</tr>
		<tr>
			<td>Control 1x106</td>
			<td>-</td>
			<td>1.9</td>
			<td>10</td>
			<td>12.3</td>
		</tr>
		<tr>
			<td>PFA fixed 0,5x106</td>
			<td>5</td>
			<td>2</td>
			<td>6.4</td>
			<td>5.5</td>
		</tr>
		<tr>
			<td>PFA  fixed 0,5x106</td>
			<td>10</td>
			<td>1.9</td>
			<td>8.1</td>
			<td>3.7</td>
		</tr>
		<tr>
			<td>PFA fixed 0,5x106</td>
			<td>15</td>
			<td>1.8</td>
			<td>8.2</td>
			<td>2.76</td>
		</tr>
		<tr>
			<td>PFA fixed 1 x106</td>
			<td>5</td>
			<td>2</td>
			<td>8.2</td>
			<td>6.26</td>
		</tr>
		<tr>
			<td>PFA fixed 1 x106</td>
			<td>10</td>
			<td>1.1</td>
			<td>8.4</td>
			<td>5.12</td>
		</tr>
		<tr>
			<td>PFA fixed 1 x106</td>
			<td>15</td>
			<td>0.9</td>
			<td>8.5</td>
			<td>5</td>
		</tr>
	</tbody>
</table>
Table 1: Quality of RNA isolated from 4% PFA fixed AECII - impact of different fixation times. RNA from AECII was isolated according to the manufacturer's protocol (FFPE RNeasy Kit) with modifications described in step 6. The fixed cells were incubated at 80 °C for either 5 min, 10 min, or 15 min to elute the RNA from the sample. RNA integrity was determined using the Fragment Analyzer from Agilent

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.

Alveolar type II epithelial cells (AECII) critically contribute to immune regulation in the lung and thus are key elements orchestrating pulmonary ...

Our research aims to identify the roles of alveolar type II epithelial cells, in short AECII, for lung development, infection and inflammation. As a novelty, our protocol enables AECII isolation from neonatal and juvenile mice, including contagious samples. To begin, obtain a euthanized juvenile mouse.After exsanguination, carefully remove the diaphragm to expose the heart and lungs. Open the rib cage using scissors. For enzymatic digestion for adult and juvenile mice, expose the trachea by removing the salivary glands and surrounding muscle.Insert a 22-gauge indwelling cannula into the trachea. Remove the needle and advance the plastic catheter toward the lungs. Secure a piece of yarn around the trachea and catheter to hold it in place.Instill dispase through the catheter to prepare the lung tissue. Exchange the syringe and instill low-melt agarose at 1%weight per volume in 45 degrees Celsius to maintain lung structure. Now, cover the lung with laboratory tissue paper and add ice for two minutes to allow the agarose to solidify.Remove the ice, tissue paper, syringe, and catheter. Then tie the yarn to close the trachea and retain the enzyme in the lower airways. Remove the heart and thymus.Cut the trachea above the yarn and remove the intact lung from the chest. Transfer the lung into a 15 milliliter tube containing pre-warmed dispase. Incubate the juvenile lung tissue in dispase at room temperature for 45 minutes.Remove the lung from dispase and dissect the lung lobes from the bronchial stem. Place the lung lobes into a Petri dish containing seven milliliters of DMEM supplemented with HEPES and 100 microliters DNase. Using forceps, disintegrate the lung tissue and incubate for 10 minutes at room temperature to ensure efficient enzymatic digestion of the lung tissue.To sort alveolar epithelial type II cells, install a 100 micrometer nozzle on the fluorescence-activated cell sorting device. Filter the antibody-labeled cells through a 50 micrometer cell strainer before sorting and rinse the strainer with supplemented DMEM to prevent cell loss. Resuspend the cells by vortexing immediately before sorting.Gate for alveolar epithelial type II cells by selecting events with high side scatter, negative lineage fluorochromes, and positive CD326 signal. Exclude doublets or aggregates using forward scatter height versus area and side scatter height versus area. Collect the sorted cells into tubes containing supplemented DMEM and centrifuge for further analysis or culture.Flow-sorting generated pure alveolar epithelial type II cell populations, with post-sort analysis validating almost 100%specificity for established cellular markers. Quantification of post-sort samples showed yields of approximately 0.15 million viable alveolar epithelial type II cells from neonatal lungs and up to 1 million cells from adult lungs per whole lung preparation. Isolated alveolar epithelial type II cells maintained typical cellular morphology after isolation.Cellular integrity of isolated alveolar epithelial type II cells was preserved throughout the isolation process. Isolated alveolar epithelial type II cells supported ex vivo infection with influenza A virus expressing the mCherry reporter protein, demonstrating viral replication over time. High-quality RNA was successfully extracted from paraformaldehyde fixed alveolar epithelial type II cells across different processing time points.The quality of extracted RNA showed a direct relationship with the duration of thermal treatment at 80 degrees Celsius during extraction. Quantitative reverse transcription PCR analysis confirmed successful amplification of ribosomal protein S9 and surfactant protein C transcripts from paraformaldehyde fixed and fresh alveolar epithelial type II cell samples. Our lab has previously identified crucial immunoregulatory roles and trained immunity of AECII during lung inflammation and infection.In general, obtaining high AECII yields and high purity at the same time is an experimental challenge. This protocol paves the way for future research on the roles of AECII during respiratory infection and postnatal lung development.

isolation-alveolar-type-ii-epithelial-cells-from-neonatal-juvenile

Research

JoVE Journal

Immunology and Infection

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium.  A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface  

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

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.

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

This video demonstrates an in vitro technique to identify immune cells residing in murine lungs. After isolating target immune cells from murine lung tissue, they are surface-labeled using a cocktail of fluorophore-tagged antibodies against specific markers on innate and adaptive immune cells. Additionally, monocytes, macrophages, and dendritic cells are labeled using an intracellular marker. The various immune cell types are then identified using flow cytometry.

An In Vitro Method to Identify Innate and Adaptive Immune Cells Residing in Murine Lungs

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Presented here is a protocol for primary human blood monocyte isolation as well as their differentiation into macrophages and dendritic cells and assembly with epithelial cells into a multicellular human lung model. Biological responses of cocultures composed of immune cells differentiated from either freshly isolated or thawed monocytes, upon exposure to proinflammatory stimuli, are compared.

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment

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

Isolation of Primary Mouse Lung Endothelial Cells

The present protocol describes human induced pluripotent stem cell-derived type 2 alveolar epithelial-like cells (iAT2s). These cells can be cultured as self-renewing spheres in 3D culture or adapted to air-liquid interface (ALI) culture.

Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells

Presented here is a reproducible, affordable, and robust method for the isolation and expansion of primary bronchial epithelial cells for long-term biobanking and the generation of differentiated epithelial cells by culture at the air-liquid interface.

Isolating Bronchial Epithelial Cells from Resected Lung Tissue for Biobanking and Establishing Well-Differentiated Air-Liquid Interface Cultures

This protocol details the isolation of live immune and non-immune populations from the mouse lung at a steady state and following influenza infection. It also provides gating strategies for identifying epithelial and myeloid cell subsets.

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

Learn a flow cytometry-based method to isolate alveolar type II epithelial cells from murine lungs at different developmental stages. This protocol enables infection studies and supports research on lung inflammation.

Isolation of Alveolar Type II Epithelial Cells from Neonatal, Juvenile, and Adult Murine Lungs Adaptable to Infectious Experimental Settings (Video) | JoVE Journal

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

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