Live-cell, nonlytic assays using the Nano-Glo Live Cell Assay System are limited in duration by degradation of the furimazine substrate. The Nano-Glo Endurazine and Vivazine Extended Live Cell Substrates provide an alternative live-cell-detection method for NanoLuc and NanoBiT luciferases enabling nonlytic assays for periods lasting several hours to days. For both substrates, a slow rate of ester hydrolysis leads to the steady release of furimazine throughout the experiment, a process catalyzed by cellular esterases. Once formed, furimazine can serve as a substrate for NanoLuc or NanoBiT luciferases. The Vivazine substrate typically shows increased brightness but also an increased rate of signal decay compared to the Endurazine substrate. The Endurazine substrate will provide the maximum signal stability but lower signal intensity compared to the other Nano-Glo Live Cell Substrates. Evaluate both substrates to determine which provides the best combination of signal intensity and stability is most suitable (e.g., Nano-Glo Extended Live Cell Substrate Trial Pack Cat.# N2590).