The lungs are in direct contact with the environment. Separated only by a thin layer of mucosa, the lung immune system is being exposed to dangers like pathogens, allergens, or pollutants. The lung dendritic cells form an elaborate network at the basolateral side of the epithelium and continuously sample antigens from the airway lumen. The conventional dendritic cells (cDCs) in the lung can be subdivided into two distinct subsets based on their ontogeny and are described to have distinct immunological functions. High-quality ex vivo isolation of these cells is required for experiments such as functional assays, transfer experiments, or transcriptomics and is crucial to further our knowledge concerning these subpopulations. In this chapter we describe a protocol for the isolation of both CD103+ and CD11b+ cDCs. In our protocol we compare different methods of cell isolation. We propose that the optimal isolation technique is based on the number of cells needed and the type of experiment that will be performed. If low cell numbers are required, simple flow cytometry-assisted cell sorting (FACS) is sufficient. In the case of high cell numbers that will be lysed or fixed upon sorting, positive selection of CD11c+ cells followed by FACS can be utilized. Purification of cDCs through gradient selection and subsequent sorting is found to be optimal for experiments that require large amount of cells for functional assays.
Lung Dendritic cells Cell isolation
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The authors would like to thank current and past members of the Bart Lambrecht laboratory who have made key contributions to the development and optimization of these procedures.
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