文献类型：期刊文献

英文题名：Analysis of redox landscapes and dynamics in living cells and in vivo using genetically encoded fluorescent sensors

作者：Zou, Yejun[1,2];Wang, Aoxue[1,2];Shi, Mei[1,2];Chen, Xianjun[1,2];Liu, Renmei[1,2];Li, Ting[1,2];Zhang, Chenxia[1,2];Zhang, Zhuo[1,2];Zhu, Linyong[3];Ju, Zhenyu[4];Loscalzo, Joseph[5];Yang, Yi[1,6];Zhao, Yuzheng[1,2]

机构：[1]East China Univ Sci & Technol, Shanghai Collaborat Innovat Ctr Biomfg Technol, State Key Lab Bioreactor Engn, Synthet Biol & Biotechnol Lab, Shanghai, Peoples R China;[2]East China Univ Sci & Technol, Sch Pharm, Shanghai Key Lab New Drug Design, Shanghai, Peoples R China;[3]East China Univ Sci & Technol, Sch Chem & Mol Engn, Inst Fine Chem, Shanghai, Peoples R China;[4]Jinan Univ, Inst Aging & Regenerat Med, Key Lab Regenerat Med, Minist Educ, Guangzhou, Guangdong, Peoples R China;[5]Harvard Med Sch, Brigham & Womens Hosp, Dept Med, Boston, MA USA;[6]Chinese Acad Sci, Shanghai Inst Biol Sci, CAS Ctr Excellence Brain Sci, Optogenet & Synthet Biol Interdisciplinary Res Ct, Shanghai, Peoples R China

年份：2018

卷号：13

期号：10

起止页码：2362

外文期刊名：NATURE PROTOCOLS

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000446087000012)】；

基金：We thank S.J. Remington for the roGFP1 vector; V.V. Belousov for the HyPer and HyPerRed vectors; J. Du for the pTol2 vector; J. Yi for the psPAX2 and pMD2. G vectors; N. Su, L. Huang, Q. Wang, P. Ni, and H. Zi for technical assistance; and S.C. Tribuna for secretarial assistance. This research was supported by the National Key Research and Development Program of China (2017YFC0906900, 2017YFA050400, 2016YFA0100602, and 2017YFA0103302), the NSFC (31722033, 91649123, 31671484, 31225008, 31470833, 91749203, 81525010, and 81420108017), the Shanghai Science and Technology Commission (14XD1401400, 16430723100, and 15YF1402600), the Young Elite Scientists Sponsorship Program by Cast, Shanghai Young Top-notch Talent, the State Key Laboratory of Bioreactor Engineering, the Fundamental Research Funds for the Central Universities, the US National Institutes of Health (HL061795, HG007690, and GM107618 to J.L.), and the American Heart Association (D700382 to J.L.).

语种：英文

摘要：Cellular oxidation-reduction reactions are mainly regulated by pyridine nucleotides (NADPH/NADP(+) and NADH/NAD(+)), thiols, and reactive oxygen species (ROS) and play central roles in cell metabolism, cellular signaling, and cell-fate decisions. A comprehensive evaluation or multiplex analysis of redox landscapes and dynamics in intact living cells is important for interrogating cell functions in both healthy and disease states; however, until recently, this goal has been limited by the lack of a complete set of redox sensors. We recently reported the development of a series of highly responsive, genetically encoded fluorescent sensors for NADPH that substantially strengthen the existing toolset of genetically encoded sensors for thiols, H2O2, and NADH redox states. By combining sensors with unique spectral properties and specific subcellular targeting domains, our approach allows simultaneous imaging of up to four different sensors. In this protocol, we first describe strategies for multiplex fluorescence imaging of these sensors in single cells; then we demonstrate how to apply these sensors to study changes in redox landscapes during the cell cycle, after macrophage activation, and in living zebrafish. This approach can be adapted to different genetically encoded fluorescent sensors and various analytical platforms such as fluorescence microscopy, high-content imaging systems, flow cytometry, and microplate readers. A typical preparation of cells or zebrafish expressing different sensors takes 2-3 d; microscopy imaging or flow-cytometry analysis can be performed within 5-60 min.