Isolation of Lung Retinoid-Containing Cells by Cell Sorting

Retinoids (vitamin A and its metabolites) are an essential lipid component of the alveolar microenvironment, and cell-type specific retinoid metabolism is required to maintain the functional health of the developing and adult lungs. Lung cells utilize specific pathways, allowing for the efficient uptake of circulating retinoids from the blood as retinol (ROH), followed by intracellular stepwise conversion of ROH into the transcriptionally active retinoid species, all-trans-retinoic acid (ATRA). ATRA-mediated (or retinoid-mediated) signaling is crucial for regulating lung alveolarization, surfactant production, angiogenesis, permeability, and immunity. Importantly, specific lung cells, including fibroblasts, can accumulate retinoids in the form of retinyl esters (RE), which can be stored or further mobilized as ROH for transfer to the neighboring cells when needed. Lung retinoid-containing cells can be isolated and collected from the single-cell suspension of digested lungs by making use of retinoid autofluorescence (the emission at 455 nm upon excitation at 350 nm) and by employing fluorescence-activated cell sorting (FACS). Additional cell-specific in vivo labeling of lung cells with red fluorescent protein allows isolating and collecting specific retinoid-containing lung cell populations. The collected cells can be directly analyzed or cultured for further analyses of cell morphology, gene expression, and responsiveness to pharmacological manipulations. This technique of isolation and application is important for animal model studies of lung health and lung injury to gain deeper insight into cellular aspects of retinoid metabolism in the lungs and lipid-mediated cellular communications.