Abe Masahiro, Nishihara Ryo, Kim Sung-Bae, Suzuki Koji
Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Japan.
Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
Methods Mol Biol. 2021;2274:103-110. doi: 10.1007/978-1-0716-1258-3_10.
Coelenterazine (CTZ) is the most general substrate for marine luciferases. The present protocol introduces a near-infrared (NIR ) bioluminescence (BL) imaging of mammalian cells with a cyanine-5 (Cy5) dye-conjugated CTZ . This unique Cy5-conjugated CTZ, named Cy5-CTZ , can act as a dual optical readout emitting both fluorescence (FL) and BL. The Cy5-CTZ exerts through-bond energy transfer (TBET)-based imaging modalities for mammalian cells. This novel derivative, Cy5-CTZ , is intrinsically fluorescent and emits NIR-shifted BL when reacting with an appropriate luciferase , such as Renilla luciferase (RLuc). The protocol exemplifies a unique live-cell imaging with Cy5-CTZ that is optically stable in physiological samples and rapidly permeabilize through plasma membrane and emit NIR-BL in live mammalian cells.
腔肠素（CTZ）是海洋荧光素酶最常用的底物。本实验方案介绍了一种用菁-5（Cy5）染料偶联的CTZ对哺乳动物细胞进行近红外（NIR）生物发光（BL）成像的方法。这种独特的偶联Cy5的CTZ，名为Cy5-CTZ，可作为一种双光学读出方式，同时发出荧光（FL）和生物发光。Cy5-CTZ对哺乳动物细胞采用基于穿键能量转移（TBET）的成像模式。这种新型衍生物Cy5-CTZ本身具有荧光性，当与适当的荧光素酶（如海肾荧光素酶（RLuc））反应时，会发出近红外偏移的生物发光。该实验方案例证了用Cy5-CTZ进行的独特活细胞成像，它在生理样品中光学稳定，能快速透过质膜，并在活的哺乳动物细胞中发出近红外生物发光。
