Protocol for real-time glycolytic monitoring in mammalian cells using confocal microscopy and HYlight, a biosensor for fructose 1,6-bisphosphate
Summary: 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 c...
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| Language: | English |
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Elsevier
2025-06-01
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| Series: | STAR Protocols |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166725001984 |
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| author | Israel Pérez-Chávez Nicolas Baeyens Daria Ezeriņa Esteban N. Gurzov Joris Messens |
| author_facet | Israel Pérez-Chávez Nicolas Baeyens Daria Ezeriņa Esteban N. Gurzov Joris Messens |
| author_sort | Israel Pérez-Chávez |
| collection | DOAJ |
| description | Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| format | Article |
| id | doaj-art-c1a6558948874f0a89f9c59f7d4e8cd1 |
| institution | DOAJ |
| issn | 2666-1667 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj-art-c1a6558948874f0a89f9c59f7d4e8cd12025-08-20T03:14:20ZengElsevierSTAR Protocols2666-16672025-06-016210379210.1016/j.xpro.2025.103792Protocol for real-time glycolytic monitoring in mammalian cells using confocal microscopy and HYlight, a biosensor for fructose 1,6-bisphosphateIsrael Pérez-Chávez0Nicolas Baeyens1Daria Ezeriņa2Esteban N. Gurzov3Joris Messens4VIB-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, BelgiumLaboratoire de Physiologie et de Pharmacologie (LAPP), Université Libre de Bruxelles (ULB), Brussels, BelgiumVIB-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; Corresponding authorSignal Transduction and Metabolism Laboratory, Université libre de Bruxelles (ULB), Brussels, Belgium; WELBIO Department, WEL Research Institute, Avenue Pasteur 6, 1300 Wavre, Belgium; Corresponding authorVIB-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; Corresponding authorSummary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166725001984Single cellMetabolismMicroscopy |
| spellingShingle | Israel Pérez-Chávez Nicolas Baeyens Daria Ezeriņa Esteban N. Gurzov Joris Messens Protocol for real-time glycolytic monitoring in mammalian cells using confocal microscopy and HYlight, a biosensor for fructose 1,6-bisphosphate STAR Protocols Single cell Metabolism Microscopy |
| title | Protocol for real-time glycolytic monitoring in mammalian cells using confocal microscopy and HYlight, a biosensor for fructose 1,6-bisphosphate |
| title_full | Protocol for real-time glycolytic monitoring in mammalian cells using confocal microscopy and HYlight, a biosensor for fructose 1,6-bisphosphate |
| title_fullStr | Protocol for real-time glycolytic monitoring in mammalian cells using confocal microscopy and HYlight, a biosensor for fructose 1,6-bisphosphate |
| title_full_unstemmed | Protocol for real-time glycolytic monitoring in mammalian cells using confocal microscopy and HYlight, a biosensor for fructose 1,6-bisphosphate |
| title_short | Protocol for real-time glycolytic monitoring in mammalian cells using confocal microscopy and HYlight, a biosensor for fructose 1,6-bisphosphate |
| title_sort | protocol for real time glycolytic monitoring in mammalian cells using confocal microscopy and hylight a biosensor for fructose 1 6 bisphosphate |
| topic | Single cell Metabolism Microscopy |
| url | http://www.sciencedirect.com/science/article/pii/S2666166725001984 |
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