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Protocol for real-time glycolytic monitoring in mammalian cells using confocal microscopy and HYlight, a biosensor for fructose 1,6-bisphosphate

par Perez Chavez, Israel ;Baeyens, Nicolas ;Ezeriņa, Daria;Gurzov, Esteban Nicolas ;Messens, Joris
Référence STAR Protocols, 6, 2, page (103792)
Publication Publié, 2025-04-23

Article révisé par les pairs

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Résumé :

Here, we present a protocol for a glycolytic stress test using HYlight, a fluorescent protein-based biosensor that specifically binds fructose 1,6-bisphosphate (FBP), a product of the rate-limiting step of glycolysis. We describe steps for HYlight delivery into cells, real-time imaging by confocal microscopy, and data analysis. Compared with other techniques, HYlight allows glycolytic activity assessment in intact cells with single-cell resolution. This protocol can be applied to studies in which glycolysis might play a role, such as cancer or diabetes research. For complete details on the use and execution of this protocol, please refer to Pérez-Chávez et al.1