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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, H 2 O 2 , 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.

Analysis of redox landscapes and dynamics in living cells and in vivo using genetically encoded fluorescent sensors