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

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

  • STAR Protoc. 2025 Jun 20;6(2):103792. doi: 10.1016/j.xpro.2025.103792.
Israel Pérez-Chávez 1,  Nicolas Baeyens 2,  Daria Ezeriņa 3,  Esteban N Gurzov 4,  Joris Messens 5
Affiliations

Affiliations

  • 1 VIB-VUB Center for Structural Biology, Vlaams Instituut Voor Biotechnologie, 1050 Brussels, Belgium; Brussels Center for Redox Biology, Vrije Universiteit Brussel, 1050 Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium; Signal Transduction and Metabolism Laboratory, Université libre de Bruxelles (ULB), Brussels, Belgium.
  • 2 Laboratoire de Physiologie et de Pharmacologie (LAPP), Université Libre de Bruxelles (ULB), Brussels, Belgium.
  • 3 VIB-VUB Center for Structural Biology, Vlaams Instituut Voor Biotechnologie, 1050 Brussels, Belgium; Brussels Center for Redox Biology, Vrije Universiteit Brussel, 1050 Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium. Electronic address: daria.ezerina@vub.be.
  • 4 Signal Transduction and Metabolism Laboratory, Université libre de Bruxelles (ULB), Brussels, Belgium; WELBIO Department, WEL Research Institute, Avenue Pasteur 6, 1300 Wavre, Belgium. Electronic address: esteban.gurzov@ulb.be.
  • 5 VIB-VUB Center for Structural Biology, Vlaams Instituut Voor Biotechnologie, 1050 Brussels, Belgium; Brussels Center for Redox Biology, Vrije Universiteit Brussel, 1050 Brussels, Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium. Electronic address: joris.messens@vub.be.
PMID: 40279243 DOI: 10.1016/j.xpro.2025.103792
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.

Keywords

Metabolism; Microscopy; Single cell.

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