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Abstract：
The leukocyte adhesion cascade governs the recruitment of circulating immune cells from the vasculature to distal sites. The initial adhesive interactions between cell surface ligands displaying sialyl-LewisX (sLeX) and endothelial E- and P-selectins serve to slow the cells down enough to interact more closely with the surface, polarize, and exit into the tissues. Therefore, precise microfluidic assays are critical in modeling how well immune cells can interact and \"roll\" on selectins to slow down enough to complete further steps of the cascade. Here, we present a systematic protocol for selectin mediated rolling on recombinant surfaces and endothelial cell monolayers on polyacrylamide gels of varying stiffness. We also describe step-by-step the protocol for setting up and performing the experiment and how to analyze and present the data collected. This protocol serves to simplify and detail the procedure needed to investigate the initial selectin-mediated interactions of immune cells with the vasculature. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparing dishes for cell rolling experiments Basic Protocol 2: Fabrication of polyacrylamide gels for cell rolling experiments Alternate Protocol 1: Protein conjugation with N6 linker Alternate Protocol 2: HUVEC culturing for monolayers Basic Protocol 3: Conducting cell rolling experiments on polyacrylamide gels Basic Protocol 4: ImageJ analysis of cell rolling movies Basic Protocol 5: Quantification of Fc site density on a surface (e.g., for Fc chimeras).
摘要：
白细胞粘附级联控制循环免疫细胞从脉管系统向远端部位的募集。显示唾液酸-LewisX（sLeX）的细胞表面配体与内皮E-和P-选择素之间的初始粘附相互作用可使细胞减慢速度，从而与表面更紧密地相互作用。极化，并进入组织。因此,精确的微流控分析对于模拟免疫细胞如何很好地相互作用和“滚动”选择素以足够慢来完成级联的进一步步骤至关重要。这里,我们提出了在不同硬度的聚丙烯酰胺凝胶上的重组表面和内皮细胞单层上选择素介导的滚动的系统方案。我们还逐步描述了设置和执行实验的协议，以及如何分析和呈现收集的数据。该方案用于简化和详细研究免疫细胞与脉管系统的初始选择素介导的相互作用所需的程序。©2024作者WileyPeriodicalsLLC出版的当前协议。基本方案1：准备用于细胞滚动实验的培养皿基本方案2：制备用于细胞滚动实验的聚丙烯酰胺凝胶替代方案1：与N6接头的蛋白质缀合替代方案2：HUVEC培养单层基本方案3：在聚丙烯酰胺凝胶上进行细胞滚动实验基本方案4：ImageJ分析细胞滚动膜基本方案5：量化表面上的Fc位点密度（例如，对于Fc嵌合体)。
