van de Laar Lianne, Guilliams Martin, Tavernier Simon
Laboratory of Immunoregulation, VIB Inflammation Research Center, Ghent University, Technologiepark 927, 9052, Ghent, Belgium.
Department of Internal Medicine, Ghent University, Ghent, Belgium.
Methods Mol Biol. 2016;1423:139-52. doi: 10.1007/978-1-4939-3606-9_10.
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.
肺直接与外界环境接触。仅隔着一层薄薄的黏膜，肺免疫系统就暴露于病原体、过敏原或污染物等危险之中。肺树突状细胞在上皮细胞基底外侧形成一个精细的网络，并持续从气道腔中摄取抗原。肺中的传统树突状细胞（cDCs）可根据其个体发育分为两个不同的亚群，且具有不同的免疫功能。对于功能分析、移植实验或转录组学等实验而言，需要高质量地离体分离这些细胞，这对于增进我们对这些亚群的了解至关重要。在本章中，我们描述了一种分离CD103(+)和CD11b(+) cDCs的方案。在我们的方案中，我们比较了不同的细胞分离方法。我们认为，最佳的分离技术取决于所需细胞数量以及将要进行的实验类型。如果需要的细胞数量较少，简单的流式细胞仪辅助细胞分选（FACS）就足够了。如果需要大量在分选后会被裂解或固定的细胞，可以采用先对CD11c(+)细胞进行阳性选择然后再进行FACS的方法。通过梯度选择和后续分选来纯化cDCs，对于需要大量细胞进行功能分析的实验而言是最佳选择。
