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

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.

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development of disease. Studies utilizing mouse models of respiratory infection, allergy, fibrosis, and cancer have been critical to reveal mechanisms of disease progression and identify therapeutic targets. However, most studies focused on the mouse lung prioritize the isolation of either immune cells or epithelial cells, rather than both populations concurrently. Here, we describe a method for preparing a comprehensive single-cell suspension of both immune and non-immune populations suitable for flow cytometry and fluorescence-activated cell sorting. These populations include epithelial cells, endothelial cells, fibroblasts, and a variety of myeloid cell subsets. This protocol entails bronchoalveolar lavage and subsequent inflation of the lungs with dispase. Lungs are then digested in a liberase mixture. This method of processing liberates a variety of diverse cell types and results in a single-cell suspension that does not require manual dissociation against a filter, promoting cell survival and yielding high numbers of live cells for downstream analyses. In this protocol, we also define gating schemes for epithelial and myeloid cell subsets in both naïve and influenza-infected lungs. Simultaneous isolation of live immune and non-immune cells is key for interrogating intercellular crosstalk and gaining a deeper understanding of lung biology in health and disease.

The lung is composed of the airways, alveoli, and interstitium. Immune and non-immune cells reside within these compartments to contribute to both homeostatic lung function (gas exchange) and host defense against environmental insults, such as viral infection. The large and small airways, or the bronchi and bronchioles, are lined by epithelial cells. The predominant epithelial cells in these regions are club and ciliated cells which are responsible for secreting protective molecules and facilitating mucociliary clearance¹. The alveoli are the most distal structures in the lung, lined by two epithelial cell types, alveolar type I cells (ATIs) an....

This protocol complies with the guidelines of the Institutional Animal Care and Use Committee at Harvard Medical School (Grant numbers: R35GM150816 and P30DK043351). Female C57BL/6J mice aged 8-12 weeks were used for the experiments. This protocol is also suitable for male mice. The details of the reagents and equipment used in this study are provided in the Table of Materials.

1. Preparation of materials

1. Thaw necessary enzymes on ice, including di.......

A successful digest will result in approximately 20-25 million cells with 90%-95% viability. If approximately 25,000 counting beads are added to an 8% fraction of the lung, beads should compromise 1%-3% of collected events. After gating on singlets, approximately 90%-95% of cells should be Zombie Aqua negative (indicating viability) (Figure 2A, Figure 3A).

Of CD45⁺ cells, CD64⁺F4/80⁺ cells are defined .......

This protocol outlines a mouse lung digest that isolates approximately 20-25 million cells per mouse with 90%-95% viability. It additionally allows for the collection of BALF for further analysis. The resultant cell suspension is compatible with multiple laboratory techniques, including flow cytometry and fluorescence-activated cell sorting to isolate cells for sequencing or cell culture. Briefly, after perfusion, BALF is collected, and lungs are inflated with dispase. Lungs are then chopped and digested in a liberase/DN.......

The authors have nothing to disclose.

This work was supported in part by grants from the National Institutes of Health (R35GM150816 and P30DK043351), Charles H. Hood Foundation, and Harvard Stem Cell Institute. We thank Alexander Mann and all other members of the Franklin laboratory for their help and advice in designing and refining the flow cytometry gating schemes and analyses. We also thank the Immunology Flow Cytometry Core at Harvard Medical School. Flow cytometry analysis was performed using FlowJo. Figure schematics were created using BioRender.