Supplemental Materials - Multi-Compartmental Analysis of the Murine Pulmonary Immune Response by Spectral Flow Cytometry

Abstract

Studies of pulmonary inflammation require unique considerations due to the complex structure and composition of the lungs. The lungs have multiple compartments, and diverse immune cell populations, with inherently high autofluorescence, and are involved in the host response to pulmonary pathogens and environmental factors. Here we describe a protocol that accounts for these factors through a novel combination of methodologies – in vivo compartmental analysis and spectral flow cytometry allowing for a broad panel of antibodies and the ability to minimize autofluorescence in immune cells. In vivo compartmental analysis enables the precise localization of immune cells within the marginated vasculature, the lung interstitium, the non-lavageable airways, and the lavageable airways of the lungs, as well as within the pulmonary lymph nodes. Spectral flow cytometry maximizes the information that can be obtained regarding the diverse leukocyte subpopulations involved in the pulmonary response. A broad panel of antibodies supports an unbiased exploratory approach to investigating diverse immune cell populations during pulmonary inflammation. Most importantly, spectral flow utilizes cellular autofluorescence to aid in the resolution and identification of immune cell populations. This methodology enables the acquisition of high-quality data compatible with informed gating and dimensionality reduction algorithms. Additionally, our protocol emphasizes considerations for compartmentalization of the inflammatory response, spectral flow panel design, and autofluorescence spectra analysis. The methodologies employed by this protocol, including an unbiased approach, are critical for increasing the rigor of pulmonary research. We apply this protocol for the precise characterization and localization of immune cells within the lungs of C57BL6/J mice during the transition from the innate to an adaptive immune response to the influenza A virus. We demonstrate that implementing this protocol improves the quantification and localization of alveolar macrophages within the airways. The methodology is modifiable and expandable to allow for further characterization of immune cell populations of particular interest. We also present considerations for applying this methodology to studies in other mouse models and with other agonists, including lipopolysaccharide, bleomycin, and house dust mite allergens.