Vajen Tanja, Heinzmann Alexandra C A, Dickhout Annemiek, Zhao Zhen, Nagy Magdolna, Heemskerk Johan W M, Koenen Rory R
Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University;
Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University.
J Vis Exp. 2018 Apr 9(134):57009. doi: 10.3791/57009.
The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.
近几十年来，人们对损伤或炎症发生时白细胞募集到损伤部位或组织损伤处的情况进行了研究，由此产生了白细胞黏附级联反应的概念。然而，白细胞募集中涉及的确切分子机制尚未完全明确。由于白细胞募集仍是感染、炎症和（自身）免疫研究领域的一个重要课题，我们提出了一种基于层流的简单检测方法，用于研究在静脉和动脉血流状态下白细胞黏附、脱黏附和迁移的潜在机制。该体外检测方法可用于研究在不同血管炎症模型中白细胞与其细胞伙伴之间相互作用的分子机制。本方案描述了一种基于层流的检测方法，使用平行流室和连接相机的倒置相差显微镜来研究白细胞与内皮细胞或血小板的相互作用，这些相互作用可以被可视化、记录，然后离线分析。内皮细胞、血小板或白细胞可以用抑制剂或抗体进行预处理，以确定特定分子在此过程中的作用。剪切条件，即动脉或静脉剪切应力，可以通过灌注液的粘度和流速以及通道高度轻松调整。
