Protocol for real-time glycolytic monitoring in mammalian cells using confocal microscopy and HYlight, a biosensor for fructose 1,6-bisphosphate
Year: 2025
Type: article
Abstract: 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 • Steps for cell seeding and HYlight transfection in cell lines • Device setup for imaging of HYlight in fluorescence microscope systems • Glycolytic stress test via fructose 1,6-bisphosphate imaging with HYlight in single cells • Analysis of fructose 1,6-bisphosphate dynamics and its correlation with glycolysis Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.
Source: STAR Protocols
Language: English
Subfield: Biophysics
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,550
Funders: Fonds De La Recherche Scientifique - FNRS, Vlaams Instituut voor Biotechnologie, Walloon excellence in life sciences and biotechnology, European Research Council, Fonds pour la Formation à la Recherche dans l’Industrie et dans l’Agriculture, H2020 European Research Council, HORIZON EUROPE European Research Council
OpenAlex ID: W4409718385