Single-Cell Flow Cytometry Profiling of BAL in Children
Shivanthan Shanthikumar, Matthew J. Burton, Richard Saffery, Sarath Ranganathan, Melanie R. Neeland
Abstract
Abstract Childhood pulmonary diseases not only cause childhood morbidity and mortality but also can cause long-term pulmonary impairment. The clinical management of many childhood pulmonary diseases is hampered by a limited understanding of the underlying pathophysiological mechanisms. Flow cytometry, which can be used to phenotype individual cell populations or isolate cells for downstream analysis, represents a crucial technology that can help to elucidate the pathophysiology of these conditions. Here, we describe a flow cytometry–based method for purification and characterization of cell populations in BAL from children. This includes assessment of the effect of cryopreservation on cell phenotype and frequency, a knowledge gap recently identified by an American Thoracic Society report on flow cytometry in lung samples. To our knowledge, this is the first study to simultaneously quantify alveolar macrophages, T cells (CD4 and CD8), B cells, natural killer cells, dendritic cells, granulocytes, and monocytes (CD16+/CD16−) in the BAL of children. The protocols described can be used to advance investigation of the pathophysiology of childhood pulmonary diseases.