Lyons-Cohen Miranda R, Thomas Seddon Y, Cook Donald N, Nakano Hideki
Immunity, Inflammation, and Disease Laboratory, Division of Intramural Research, National Institute of Environmental Health Sciences, NIH.
Immunity, Inflammation, and Disease Laboratory, Division of Intramural Research, National Institute of Environmental Health Sciences, NIH;
J Vis Exp. 2017 Apr 5(122):55465. doi: 10.3791/55465.
Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly, chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.
吸入变应原和病原体可引起肺内多种免疫细胞类型发生多种变化。流式细胞术是一种用于定量分析免疫细胞表面蛋白的强大技术，但它无法提供这些细胞在肺内的定位和迁移模式的信息。同样，可以进行趋化性测定以研究细胞在体外对趋化因子作出反应的潜力，但这些测定无法重现完整肺的复杂环境。与上述技术不同，通过制备精密肺切片（PCLS），用市售的荧光标记抗体对其进行染色，并通过共聚焦显微镜观察切片，可以很容易地观察到肺内单个细胞类型的位置。PCLS可用于活的和固定的肺组织，切片可涵盖整个肺叶横截面那么大的区域。我们已经使用该方案成功地观察到肺内多种细胞类型的位置，包括不同类型的树突状细胞、巨噬细胞、中性粒细胞、T细胞和B细胞，以及诸如淋巴管、内皮和上皮细胞等结构细胞。在活的三维肺组织中观察细胞间相互作用（如树突状细胞与T细胞之间的相互作用）的能力，可以揭示细胞在肺内如何移动以及在稳态和炎症期间如何相互作用。因此，当与其他程序（如流式细胞术和定量PCR）结合使用时，PCLS有助于全面了解肺过敏性和炎症性疾病背后的细胞事件。
