Protocol to phenotype and quantify mycobacteria-specific myeloid cells from human airways by mass cytometry

Alveolar macrophages and other myeloid cells in the human airways are the primary cell types responding to respiratory pathogens. Here, we present a protocol for in vitro stimulation of cryopreserved human bronchoalveolar lavage (BAL) cells with mycobacterial antigens for phenotyping and quantifying proinflammatory cytokine responses in myeloid cells by mass cytometry. We demonstrate that the measure of markers of myeloid lineage and function is stable after freezing stained cells thereby allowing for batched analyses and/or machine downtime. [Display omitted] •Functional and phenotypic characterization of human alveolar macrophages•Steps for thawing and stimulation of cryopreserved bronchoalveolar lavage cells•Steps for staining of bronchoalveolar lavage cells after antigen stimulation•Optimized antibody panel for mass cytometry Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Alveolar macrophages and other myeloid cells in the human airways are the primary cell types responding to respiratory pathogens. Here, we present a protocol for in vitro stimulation of cryopreserved human bronchoalveolar lavage (BAL) cells with mycobacterial antigens for phenotyping and quantifying proinflammatory cytokine responses in myeloid cells by mass cytometry. We demonstrate that the measure of markers of myeloid lineage and function is stable after freezing stained cells thereby allowing for batched analyses and/or machine downtime.