Cernuda-Morollón Eva, Gharbi Severine, Millán Jaime
Unidad de Histocompatibilidad, Hospital Universitario Central de Asturias, Oviedo, Spain.
Methods Mol Biol. 2010;616:69-82. doi: 10.1007/978-1-60761-461-6_5.
Leucocyte transendothelial migration (TEM) or diapedesis is pivotal in leucocyte trafficking during the inflammatory and immune responses. The endothelium plays an active role in this process, triggering an array of signalling pathways and reorganizing its cytoskeleton and membrane to facilitate leucocyte TEM. Diapedesis can occur between endothelial cells (paracellular) or through individual endothelial cells (transcellular). This latter route accounts for up to 30% of the total diapedesis in certain endothelial cell types in vitro. Mechanisms underlying both routes of diapedesis have been subjected to intense investigation during recent years. Here we describe a method to discriminate between the paracellular and the transcellular routes of diapedesis in vitro. The method is based on a transmigration assay of human T lymphoblasts through TNF-alpha-stimulated human primary endothelial monolayers, a triple fluorescence labelling of F-actin, the adhesion receptor ICAM-1 and the junctional protein beta-catenin and a subsequent acquisition of z-stacks of high-resolution confocal sections.
白细胞跨内皮迁移（TEM）或渗出在炎症和免疫反应期间的白细胞运输中起关键作用。内皮细胞在此过程中发挥积极作用，触发一系列信号通路并重组其细胞骨架和细胞膜以促进白细胞TEM。渗出可在内皮细胞之间（旁细胞途径）或通过单个内皮细胞（穿细胞途径）发生。在体外，后一种途径在某些内皮细胞类型的总渗出中占比高达30%。近年来，这两种渗出途径的潜在机制受到了深入研究。在此，我们描述一种在体外区分旁细胞和穿细胞渗出途径的方法。该方法基于人T淋巴母细胞通过肿瘤坏死因子-α刺激的人原代内皮单层的迁移试验、F-肌动蛋白、黏附受体ICAM-1和连接蛋白β-连环蛋白的三重荧光标记以及随后获取高分辨率共聚焦切片的z轴堆栈。
