Moran Imogen, Phan Tri Giang
Immunology Division, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, NSW, 2010, Australia.
Faculty of Medicine, St Vincent's Clinical School,, UNSW Australia, Darlinghurst, NSW, 2010, Australia.
Methods Mol Biol. 2017;1623:59-72. doi: 10.1007/978-1-4939-7095-7_5.
The germinal center (GC) reaction is the key process for the generation of high affinity antibodies to foreign antigen. Standard experimental techniques such as fluorescence-activated cell sorting and histology have provided numerous insights into the composition and function of the GC. However, these approaches are limited to a "snapshot" in time and are unable to fully capture the dynamic nature of the GC. Intravital two-photon microscopy overcomes these disadvantages and has led to several major advances in the field but is restricted by practical and technical limits that prevent long-range mapping and molecular studies. Here we describe procedures for optical marking or "tagging" of cells in precise microanatomical compartments by two-photon photoconversion that can be used for long-term fate mapping and transcript profiling of GC T and B cells.
生发中心（GC）反应是产生针对外来抗原的高亲和力抗体的关键过程。诸如荧光激活细胞分选和组织学等标准实验技术已经为生发中心的组成和功能提供了诸多见解。然而，这些方法仅限于某个时间点的“快照”，无法完全捕捉生发中心的动态特性。活体双光子显微镜克服了这些缺点，并在该领域带来了几项重大进展，但受到实际和技术限制，阻碍了远距离绘图和分子研究。在这里，我们描述了通过双光子光转化在精确的微解剖隔室中对细胞进行光学标记或“标记”的程序，该程序可用于生发中心T细胞和B细胞的长期命运图谱绘制和转录谱分析。
