Nowosad Carla R, Tolar Pavel
Laboratory of Activation of Immune Receptors, Francis Crick Institute, Mill Hill Laboratory, London, UK.
Division of Immunology and Inflammation, Department of Medicine, Imperial College London, NW7 1AA, London, UK.
Methods Mol Biol. 2017;1584:77-88. doi: 10.1007/978-1-4939-6881-7_6.
Surrogate planar and membrane systems have been employed to study the architecture of immune synapses; however, they often do not recapitulate trans-synaptic extraction and endocytosis of ligands by the immune cells. Transendocytosis (or trogocytosis) of antigen from immune synapses is particularly critical for antigen processing and presentation by B cells. Here we describe a protocol for preparation of plasma membrane sheets (PMSs), which are flexible and fluid membrane substrates that support robust B cell antigen extraction. We show how to attach B cell antigens to the PMSs and how to investigate antigen extraction and endocytosis by fluorescent microscopy and computational image analysis. These techniques should be broadly applicable to studies of transendocytosis in a variety of cellular systems.
替代平面和膜系统已被用于研究免疫突触的结构；然而，它们往往无法重现免疫细胞对配体的跨突触提取和内吞作用。免疫突触对抗原的转胞吞作用（或咬尾作用）对于B细胞的抗原加工和呈递尤为关键。在这里，我们描述了一种制备质膜片（PMS）的方案，质膜片是灵活且具有流动性的膜底物，可支持强大的B细胞抗原提取。我们展示了如何将B细胞抗原附着到质膜片上，以及如何通过荧光显微镜和计算图像分析来研究抗原提取和内吞作用。这些技术应广泛适用于各种细胞系统中转胞吞作用的研究。
