A 14-Color Blue-Violet Laser Restricted Full Spectrum Flow Cytometry Panel for Comprehensive Immunophenotyping of Pulmonary Inflammation in Mouse Bronchoalveolar Lavage Fluid

Here we describe a 16-parameter, 14-color surface staining panel optimized for murine bronchoalveolar lavage cells that enables reproducible identification of major innate and adaptive immune populations relevant to pulmonary infections or other inflammatory conditions of the airways. The panel enables confident identification of neutrophils, eosinophils, B cells, T cells and subtypes, NK cells, and distinguishes between tissue resident and monocyte-derived macrophages populations. The panel was carefully designed for BAL samples that vary in cell number, are rich in debris, and often autofluorescent. Antibody concentrations are optimized to provide reproducible results regardless of sample-variable cell numbers allowing for the preparation of a single antibody cocktail master mix and rapid sample staining time, thereby cutting down on sample preparation and optimizing cell viability of analyzed samples. The panel facilitates robust cross-sectional and longitudinal comparison of airway inflammation across different airway inflammatory conditions, infections by different respiratory pathogens, impact of vaccination or therapeutics on the inflammatory landscape, and more. It facilitates hypothesis generation by revealing recruitment kinetics and remodeling of myeloid compartments, supports downstream sorting for transcriptomic or functional assays, and provides a standardized baseline for labs to adopt or extend for activation or intracellular cytokine analyses. We have successfully utilized this panel to identify differential host responses to different respiratory Mycoplasma pathogens as well as to longitudinally track the progression of inflammatory response to Mycoplasma pneumoniae over a 21-day time course study. This panel provides an economic immunophenotyping option by utilizing only 14 markers and a two laser (Blue and Violet) full spectrum cytometer to provide comprehensive immunophenotyping power of both myeloid and lymphoid cells. Furthered by lacking the requirement for advanced unmixing for sample analysis, the panel can be easily adopted by the community, enabling comparative meta-analyses of host responses across murine respiratory infection models.