Imaging the Human Immunological Synapse
Imaging the Human Immunological Synapse
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DOI：
10.3791/60312
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发表时间：
2019-12-01
影响因子：
1.2
通讯作者：
Izquierdo, Manuel
中科院分区：
文献类型：
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作者：
Bello-Gamboa, Ana;Manuel Izquierdo, Juan;Izquierdo, Manuel
该方法的目的是生成免疫突触(IS)，这是由抗原提呈细胞(APC)和效应辅助T淋巴细胞(Th)形成的细胞间结合的例子，并记录对应于IS形成的第一阶段和随后的贩运事件(发生在APC和Th细胞中)的图像。这些事件最终将导致IS的两极分泌。在该方案中，由于该实验系统接近生物现实(Th细胞-APC突触结合物)，使用葡萄球菌肠毒素E(SEE)冲击的Raji细胞作为细胞突触模型。本文介绍的方法包括细胞间共轭、延时采集、广场荧光显微镜(WFFM)和图像处理(采集后去卷积)。这改善了图像的信噪比(SNR)，提高了时间分辨率，允许同步获取新出现的突触共轭中的几个荧光染料，并减少了荧光漂白。此外，该方案与终点细胞固定方案(多聚甲醛、丙酮或甲醇)很好地匹配，这将允许进一步的免疫荧光染色和分析。该方案还兼容激光扫描共聚焦显微镜(LSCM)和其他最先进的显微镜技术。作为主要警告，只有那些沿Z轴与焦点平面成90度角的T细胞-APC边界(称为IS界面)才能被正确成像和分析。存在其他实验模型，简化了Z维成像和后续图像分析，但这些方法不能模拟APC复杂、不规则的表面，并可能促进IS中的非生理相互作用。因此，这里使用的实验方法适合于复制和对抗IS中发生的一些生物复杂性。
The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90 degrees angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.