Contreras Osvaldo, Thekkedam Chris, Zaunders John, Aguirre-MacLennan Ismael, Murray Nicholas J, Gonzalez-Cordero Anai, Harvey Richard P
Victor Chang Cardiac Research Institute, Darlinghurst, NSW 2010, Australia.
School of Clinical Medicine, Faculty of Medicine & Health, UNSW Sydney, Kensington, NSW 2052, Australia.
iScience. 2025 Aug 16;28(9):113380. doi: 10.1016/j.isci.2025.113380. eCollection 2025 Sep 19.
5-Ethynyl-2'-deoxyuridine (EdU) has revolutionized DNA replication and cell cycle analyses through fast, efficient click chemistry detection. However, commercial EdU kits suffer from high costs, proprietary formulations, limited antibody multiplexing capabilities, and difficulties with larger biological specimens. Here, we present OpenEMMU (Open-source EdU Multiplexing Methodology for Understanding DNA replication dynamics), an optimized, affordable, and user-friendly click chemistry platform utilizing off-the-shelf reagents. OpenEMMU enhances efficiency, brightness, and multiplexing capabilities of EdU staining with both non-conjugated and conjugated antibodies across diverse cell types, including T cell activation and proliferation assays. We validated its effectiveness for the fluorescent imaging of nascent DNA synthesis in developing embryos and organs, including embryonic heart, forelimbs, and 3D hiPSC-derived cardiac organoids. OpenEMMU also enabled the deep-tissue 3D imaging of DNA synthesis in zebrafish larvae and under replication stress in embryos at high spatial resolution. This approach opens new avenues for understanding organismal development, cell proliferation, and DNA replication dynamics with unprecedented precision and flexibility.
5-乙炔基-2'-脱氧尿苷（EdU）通过快速、高效的点击化学检测方法，彻底改变了DNA复制和细胞周期分析。然而，商业化的EdU试剂盒存在成本高、配方专有、抗体多重标记能力有限以及在处理较大生物样本时存在困难等问题。在此，我们介绍OpenEMMU（用于理解DNA复制动力学的开源EdU多重标记方法），这是一个利用现成试剂的优化、经济且用户友好的点击化学平台。OpenEMMU通过非共轭和共轭抗体，提高了不同细胞类型（包括T细胞活化和增殖检测）中EdU染色的效率、亮度和多重标记能力。我们验证了其在发育中的胚胎和器官（包括胚胎心脏、前肢和3D人诱导多能干细胞衍生的心脏类器官）中对新生DNA合成进行荧光成像的有效性。OpenEMMU还能够在高空间分辨率下对斑马鱼幼虫的DNA合成进行深层组织3D成像，并对处于复制应激状态的胚胎进行成像。这种方法为以前所未有的精度和灵活性理解生物体发育、细胞增殖和DNA复制动力学开辟了新途径。
