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

Summary

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.

Abstract

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 × 10⁶ AECII cells per adult lung, this protocol supports preclinical studies of, e.g., viral pathogenesis, epithelial function, and regenerative capacity.

Introduction

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, age^2,3) 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 AECII^5,6. 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.

Protocol

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 naïve mice for subsequent AECII isolation was performed in accordance with national guidelines from the Society of Laboratory Animal Science (GV-SOLAS, Gesellschaft für 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

1. Sacrifice the mouse (following institutionally approved protocols):
   1. For adult and juvenile mice: administer an anesthesia overdose (e.g., ketamine/xylazine) via intraperitoneal injection.
   2. For neonatal mice: decapitate.
      NOTE: Avoid cervical dislocation in adult and juvenile mice as this procedure can damage the trachea.
2. 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.
3. Carefully remove the skin and peritoneum.
4. Exsanguinate the mouse by cutting both jugular veins and the vena cava, allowing blood to drain from the body.
5. 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.
6. Open the ribcage using scissors.
7. 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:
   1. For adult mice: use a 26 G cannula and perfuse the lung with 10 mL PBS.
   2. 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.

2. Enzymatic digestion of lung tissue

1. Adult and juvenile mice:
   1. Expose the trachea by removing the salivary glands and the surrounding muscle.
   2. Insert a 22 G indwelling cannula into the trachea, remove its needle, and move the plastic catheter towards the lungs.
   3. Securely tie a piece of yarn around both trachea and catheter to hold it in place.
   4. 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:
      1. For adult mice: Insert 2 mL of dispase.
      2. For juvenile mice: Insert 500 µL 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.
   5. Exchange the syringe and repeat with low-melt agarose (1% (w/v), 45 °C) to maintain structure during processing.
      1. For adult mice: Use 500 µL of low-melt agarose.
      2. For juvenile mice: Use 200 µL 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 °C), let agarose solution cool down to 45 °C, e.g., in a thermoblock.
   6. Cover the lung with laboratory tissue paper and add ice to allow the agarose to solidify for 2 min.
   7. Remove ice, tissue paper, syringe, and catheter.
   8. Tie the yarn to close the trachea in order to keep the enzyme in the lower airways.
   9. Remove heart and thymus.
   10. Cut the trachea above the yarn and remove the intact lung from the chest.
   11. Transfer the lung into a 15 mL tube containing pre-warmed dispase (37 °C) and incubate the lung tissue in dispase for 45 min at room temperature (20-25 °C).
       1. For adult mice: incubate the lung in 2 mL dispase.
       2. For juvenile mice: incubate the lung in 1.5 mL dispase.
2. Neonatal mice
   1. Collect and mince single lung lobes using fine scissors in the lid of a 2 mL tube.
   2. Add 500 µL dispase, close the lid, and invert the tube to ensure that the entire tissue is covered in dispase.
   3. Incubate the lung tissue in dispase at room temperature for 45 min.

3. Preparation of the lung tissue

1. For adult and juvenile mice:
   1. Remove the lung from the dispase and dissect the lung lobes from the bronchial stem.
   2. Place the lung lobes into a Petri dish (diameter: 8.5 cm) containing 7 mL DMEM + HEPES, 1 µg/mL (final concentration) anti-CD16/32 blocking antibody and 100 µL 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.
   3. 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.
   4. Incubate for 10 min at room temperature while gently rocking at 200 rpm.
      NOTE: Cell suspension can be stored at 4 °C until the next step, if required.
2. For neonatal mice:
   1. Transfer the dispase, including the digested tissue, to a Petri dish (diameter: 5.4 cm) containing 3 mL DMEM + HEPES, 1 µg/mL (final concentration) anti-CD16/32 blocking antibody and 100 µL DNAse (final concentration ~ 600 Kunitz units).
   2. Incubate for 10 min at room temperature while gently rocking at 200 rpm.
      NOTE: Cell suspension can be stored at 4 °C until the next step, if required.
3. Continue for all age groups:
   1. Filter the cell suspension through a series of nylon cell strainers with decreasing pore size (100 µm - 70 µm - 40 µm) to remove debris and large aggregates.
   2. 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.
   3. 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 °C.
   4. Discard the supernatant and resuspend the cell pellet in erythrocyte lysis buffer (ACK buffer).
      1. For adult and juvenile mice: Use 2 mL of erythrocyte lysis buffer and incubate for 2 min.
      2. For neonatal mice: Use 3 mL of erythrocyte lysis buffer and incubate for 3 min.
   5. 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 °C.

4. Antibody staining for fluorescence-activated cell sorting

1. 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.
   1. For adult mice: Use 600 µL antibody solution per lung.
   2. For juvenile mice: Use 400 µL antibody solution per lung.
   3. For neonatal mice: Use 200 µL antibody solution per lung.
2. Incubate the cell suspension with the antibodies for 10 min at 4 °C in the dark.
3. After incubation, wash the cells by filling the tube with DMEM + HEPES + 10% (v/v) FBS.
4. Centrifuge for 12 min at 140 x g at 4 °C.
5. Resuspend the cells DMEM + HEPES + 10% (v/v) FBS:
   1. For adult mice: Resuspend in 300 µL per lung.
   2. For juvenile mice: Resuspend in 200 µL per lung.
   3. For neonatal mice: Resuspend in 100 µL per lung.

5. Sorting of AECII

NOTE: Instrument preparation: install a 100 µm nozzle for sorting AECII using a FACS device.

1. Directly prior to sorting, filter cells through a 50 µm cell strainer. Rinse the cell strainer with DMEM supplemented with HEPES and 10% (v/v) FBS to prevent excessive cell loss:
   1. Adult mice: Rinse the cell strainer with 300 µL per lung.
   2. Juvenile mice: Rinse the cell strainer with 200 µL per lung.
   3. Neonatal mice: Rinse the cell strainer with 100 µL per lung.
2. Resuspend the cells by vortexing directly before sorting.
3. Gate for AECII by identifying events that I) have high side scatter (SSC^high), 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, SSC^high, and autofluorescence^{positive/high} cells. AECII autofluorescence can be identified in green light detectors, e.g., FITC or GFP detectors.
4. 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).
5. Collect the sorted cells into a tube containing DMEM supplemented with HEPES and 10% (v/v) FBS and centrifuge for further analysis or culture.

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 pathogens^10,11 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.

1. For inactivation of viruses, fix antibody-labeled single cell suspensions in 500 µL 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.
2. After fixation, wash cells with DMEM and centrifuge for 15 min at 140 x g.
3. Resuspend the cells in 300 µL DMEM + HEPES + 10% (v/v) FBS and pass the cell suspension through a 100 µm cell strainer.
4. Rinse the cell strainer with an additional 300 µL DMEM + HEPES + 10% (v/v) FBS medium to prevent excessive cell loss.
5. Sort cells as described in step 5. "Sorting of AECII".
   NOTE: After mandatory experimental verification that any virus was inactivated by treatment with 4% (w/v) PFA, AECII can be sorted under BSL1 conditions.
6. 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:
   1. After sorting, centrifuge the cells for 15 min at 140 x g at 4 °C.
   2. Resuspend the cells in 150 µL Buffer PKD and transfer into a 1.5 mL tube.
   3. Add 10 µL of Proteinase K and incubate at 56 °C for 15 min, followed by incubation at 80 °C for up to 15 min, depending on the cell count (see Table 1).
7. Continue with the RNA isolation process as per the protocol, including DNase treatment, washing steps, and final elution in 30 µL RNase-free water.

Results

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 (>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).

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 SSC^high profile of granular AECII. This protocol enables the isolation of highly pure (≥ 95%), viable primary AECII for downstream functional and molecular analyses. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

Figure 6: Amplification curves of the Rps9 housekeeping gene and AECII-specific gene Sftpc from control (untreated) and 4% PFA fixed AECII. Please click here to view a larger version of this figure.

Sample	Incubation time at 80 °C	28S/18S	RQN	RNA conc. [ng/µ]
Control 0,5x106	-	2	10	5.8
Control 1x106	-	1.9	10	12.3
PFA fixed 0,5x106	5	2	6.4	5.5
PFA  fixed 0,5x106	10	1.9	8.1	3.7
PFA fixed 0,5x106	15	1.8	8.2	2.76
PFA fixed 1 x106	5	2	8.2	6.26
PFA fixed 1 x106	10	1.1	8.4	5.12
PFA fixed 1 x106	15	0.9	8.5	5

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

Discussion

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 × 10⁶ viable AECII per neonatal lung and up to 1.0 × 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 physiological^17,18,19 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., IAV^20,21 and SARS-CoV-2^22,23,24.

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 immunity^25,26, 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 >1 and RNA quality number (RQN) values >8. It is important to note that the 80 °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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ACK buffer	-	-	8.29 g NH4Cl, 1 g KHCO3, 200 µL of 0. 5 M EDTA, add 1000 mL Milli-Q, adjust pH to 7.2 to 7.4
anti-mouse CD11b	eBioscience	12-0112-83	clone M1/70; PE coupled
anti-mouse CD11c	Biolegend	117310	clone N418; APC coupled
anti-mouse CD16/32	BD Biosciences	553141	clone 93; purified
anti-mouse CD16/32	BD Biosciences	561727	clone 2.4G2; PE coupled
anti-mouse CD19	Biolegend	115508	clone 6D5; PE coupled
anti-mouse CD24	Biolegend	101808	clone M1/69; PE coupled
anti-mouse CD31	Biolegend	102408	clone 390; PE coupled
anti-mouse CD326	Biolegend	118245	clone G8.8; BV785 coupled
anti-mouse CD45	BD Biosciences	553081	clone 30-F11; PE coupled
anti-mouse CD93	eBioscience	17-5892-82	clone AA4-1; APC coupled
anti-mouse F4/80	Biolegend	123116	clone BM8; APC coupled
anti-mouse SPC	abcam	ab211326	host species: rabbit; clone: EPR19839; purified
Aria-Fusion	BD Biosciences		Cell Sorter
Aria-II SORP	BD Biosciences		Cell Sorter
Biozym Plaque Agarose	Biozym	840101	1 % (w/v) in H2O
Cell strainer (100 µm, 70 µm, 40 µm)	BD Falcon	352360, 352350, 352340
CellTrics (50 µm)	Sysmex	04-004-2327
Deoxyribonuclease I from bovine pancreas, 2000 Kunitz units/vial	Sigma-Aldrich	D4263	freshly dissolve content of 1 vial in 330 µL DMEM
Dispase, 100 mL (5000 caseinolytic units)	Corning	354235	perform sterile filtration (pore size 0.22 µm), prepare 4 mL aliquots in 15 mL tubes, store at -20 °C
DMEM	Gibco	31885-049	low glucose, with pyruvate, add HEPES (final concentration of 25 mM)
FACS Diva	BD Biosciences		Sorting Software
FlowJo			Evaluation Software for FACS data
goat anti-rabbit			Alexa 594 coupled
Indwelling cannula Introcan 22G	Braun	4252098B
RNeasy FFPE Kit	Qiagen	73504
Symphony S6 SE	BD Biosciences		Cell Sorter