Šuštar Vid, Vainio Marika, Mattila Pieta K
Institute of Biomedicine, Unit of Pathology, and MediCity Research Laboratories, University of Turku, Turku, Finland.
Methods Mol Biol. 2018;1707:243-257. doi: 10.1007/978-1-4939-7474-0_18.
The formation of the immunological synapse upon B cell activation critically depends on the rearrangement of the submembranous actin cytoskeleton. Polymerization of actin monomers into filaments provides the force required for B cell spreading on the antigen-presenting cell (APC). Interestingly, the actin network also participates in cellular signaling at multiple levels. Fluorescence microscopy plays a critical role in furthering our understanding of the various functions of the cytoskeleton, and has become an important tool in the studies on B cell activation. The actin cytoskeleton can be tracked in live cells with various fluorescent probes binding to actin, or in fixed cells typically with phalloidin staining. Here, we present the usage of TIRF microscopy and an image analysis workflow for studying the overall density and organization of the actin network upon B cell spreading on antigen-coated glass, a widely used model system for the formation of the immunological synapse.
B细胞活化时免疫突触的形成关键取决于膜下肌动蛋白细胞骨架的重排。肌动蛋白单体聚合成丝提供了B细胞在抗原呈递细胞（APC）上铺展所需的力。有趣的是，肌动蛋白网络还在多个层面参与细胞信号传导。荧光显微镜在加深我们对细胞骨架各种功能的理解方面发挥着关键作用，并且已成为B细胞活化研究中的重要工具。可以使用与肌动蛋白结合的各种荧光探针在活细胞中追踪肌动蛋白细胞骨架，或者通常用鬼笔环肽染色在固定细胞中追踪。在这里，我们介绍了全内反射荧光（TIRF）显微镜的使用方法以及一种图像分析流程，用于研究在抗原包被的玻璃上B细胞铺展时肌动蛋白网络的整体密度和组织情况，这是一种广泛用于免疫突触形成的模型系统。
