Singapore Immunology Network (SIgN), A*STAR (Agency for Science, Technology and Research), Singapore, Singapore.
Department of Microbiology, Immunology Programme, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
Methods Mol Biol. 2021;2308:177-202. doi: 10.1007/978-1-0716-1425-9_15.
The complex bone marrow microenvironment or niche is an important anatomical structure responsible for hematopoiesis and providing support to the immune cells function. Being the source of immune and blood cells, the interaction of these hematopoietic stem and progenitor cells with the cellular niches regulates their ability for self-renewal, proliferation, and differentiation. Dynamic imaging not only provides spatiotemporal information of cell motility but also the morphological changes due to cell-cell interactions in the bone marrow, providing insights into the ongoing physiological activities within the tissue. Here, we describe customized stages with compatible equipment best suited for the upright two-photon microscope, accompanied by detailed methods for both calvarial and tibial intravital imaging. We demonstrate a general protocol for calvarial imaging using a minimally invasive surgical approach, and introduce a bone shaving-based tibial imaging as a complementary method. To demonstrate the applicability of our method we used Lyz2-EGFP transgenic mice to track bone marrow neutrophil activities as an example.
复杂的骨髓微环境或龛位是一个重要的解剖结构，负责造血，并为免疫细胞的功能提供支持。作为免疫细胞和血细胞的来源，这些造血干细胞和祖细胞与细胞龛的相互作用调节它们自我更新、增殖和分化的能力。动态成像不仅提供了细胞迁移的时空信息，还提供了由于骨髓中细胞-细胞相互作用而导致的形态变化，深入了解组织内正在进行的生理活动。在这里，我们描述了与直立双光子显微镜兼容的定制阶段，以及颅骨和胫骨活体成像的详细方法。我们展示了一种使用微创外科方法进行颅骨成像的一般方案，并介绍了一种基于骨削的胫骨成像作为补充方法。为了证明我们方法的适用性，我们以追踪骨髓中性粒细胞活性为例，使用了 Lyz2-EGFP 转基因小鼠。
