Takeda Akira, Miyasaka Masayuki, Umemoto Eiji
MediCity Research Laboratory, University of Turku, Turku, Finland.
Interdisciplinary Program for Biomedical Sciences, Institute of Academic Initiatives, Osaka University, Suita, Osaka, Japan.
Methods Mol Biol. 2018;1763:43-52. doi: 10.1007/978-1-4939-7762-8_5.
T-cell motility is essential for the T cells' ability to scan antigens within lymph nodes and initiate contact with antigen-presenting cells. While T-cell migration has been extensively studied using in vitro migration assays, accumulating evidence indicates that the T-cell migration within lymph nodes is modulated by the surrounding cells and extracellular matrix, which form the confined architecture of the lymph nodes. Therefore, to understand the mechanisms of T-cell motility in vivo, their cell migration must be analyzed under physiological conditions. To this end, two-photon microscopy is extremely useful; this technique enables the tracking of fluorescently labeled cells in vivo and ex vivo, with high spatial and temporal resolutions. Here we describe the experimental procedures for applying two-photon microscopy to the in vivo and ex vivo imaging of T-cell migration in mouse lymph nodes. These approaches provide physiological insight into the mechanisms of T-cell behavior at a single-cell level in the three-dimensional lymph node environment.
T细胞的运动能力对于其在淋巴结内扫描抗原并启动与抗原呈递细胞接触的能力至关重要。虽然使用体外迁移试验对T细胞迁移进行了广泛研究，但越来越多的证据表明，淋巴结内的T细胞迁移受到周围细胞和细胞外基质的调节，这些细胞和细胞外基质构成了淋巴结的受限结构。因此，为了了解体内T细胞运动的机制，必须在生理条件下分析它们的细胞迁移。为此，双光子显微镜非常有用；该技术能够在体内和体外以高空间和时间分辨率追踪荧光标记的细胞。在这里，我们描述了将双光子显微镜应用于小鼠淋巴结内T细胞迁移的体内和体外成像的实验程序。这些方法在三维淋巴结环境中为单细胞水平上T细胞行为的机制提供了生理学见解。
