Sauer Kerstin A, Scholtes Petra, Karwot Roman, Finotto Susetta
Laboratory of Cellular and Molecular Immunology of the Lung, University of Mainz, Germany.
Nat Protoc. 2006;1(6):2870-5. doi: 10.1038/nprot.2006.435.
The regulation of the cellular immune response in lung diseases is not yet fully understood. Isolating different subsets of immune cells directly from the lung is therefore an indispensable method of gaining detailed knowledge on the function of these cells in this organ. This protocol describes a method of isolating and magnetically labeling CD4+ lung T cells, which are then loaded and retained on the column while all other cells run through it (positive selection). The yield of this isolation is approximately 5 x 10(5) to 1.5 x 10(6) CD4+ cells from a murine lung. These cells can be further investigated by several methods such as flow cytometry, western blot analysis, RT-PCR, immunostaining and ELISA. In addition, lung CD4+ T cells alone or along with other immunologically important cells such as CD8+ T cells and T regulatory cells can be adoptively transferred into immune-deficient mice, and can influence important local parameters. This protocol can be completed in approximately 4 h 20 min.
肺部疾病中细胞免疫反应的调控尚未完全明确。因此，直接从肺部分离不同亚群的免疫细胞是深入了解这些细胞在该器官中功能的不可或缺的方法。本方案描述了一种分离和磁性标记CD4+肺T细胞的方法，随后这些细胞被加载并保留在柱上，而所有其他细胞则通过柱子（阳性选择）。从一只小鼠肺中分离得到的CD4+细胞产量约为5×10⁵至1.5×10⁶个。这些细胞可以通过多种方法进一步研究，如流式细胞术、蛋白质印迹分析、逆转录聚合酶链反应、免疫染色和酶联免疫吸附测定。此外，单独的肺CD4+ T细胞或与其他免疫重要细胞如CD8+ T细胞和T调节细胞一起，可以过继转移到免疫缺陷小鼠体内，并能影响重要的局部参数。本方案大约可在4小时20分钟内完成。
