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.

中文翻译：

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使用遗传编码的荧光传感器分析活细胞和体内的氧化还原态势和动力学。

细胞氧化还原反应主要受吡啶核苷酸（NADPH / NADP ⁺和NADH / NAD ⁺），硫醇和活性氧（ROS）调控，在细胞代谢，细胞信号传导和细胞命运决定中起着重要作用。对完整的活细胞中氧化还原态势和动力学的全面评估或多重分析，对于询问健康和疾病状态下的细胞功能都很重要。然而，直到最近，由于缺乏一套完整的氧化还原传感器，这一目标一直受到限制。我们最近报道了针对NADPH的一系列高响应性，遗传编码荧光传感器的开发，该传感器大大增强了现有的硫醇，H ₂ O ₂遗传编码传感器的工具集。，以及NADH的氧化还原状态。通过结合具有独特光谱特性和特定亚细胞靶向域的传感器，我们的方法可以同时对多达四个不同的传感器进行成像。在此协议中，我们首先描述在单个细胞中对这些传感器进行多重荧光成像的策略；然后，我们演示了如何应用这些传感器来研究细胞周期，巨噬细胞激活后和活斑马鱼中氧化还原景观的变化。这种方法可以适用于不同的基因编码的荧光传感器和各种分析平台，例如荧光显微镜，高内涵成像系统，流式细胞仪和酶标仪。表达不同传感器的细胞或斑马鱼的典型制备过程需要2-3 d。显微镜成像或流式细胞仪分析可在5-60分钟内完成。