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
  • 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: [email protected].
  • 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: [email protected].
  • 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: [email protected].
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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