Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM
We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase...
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| Format: | Article |
| Language: | English |
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MDPI AG
2016-08-01
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| Series: | Sensors |
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| Online Access: | http://www.mdpi.com/1424-8220/16/8/1312 |
| _version_ | 1818007957084831744 |
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| author | George Chennell Robin J. W. Willows Sean C. Warren David Carling Paul M. W. French Chris Dunsby Alessandro Sardini |
| author_facet | George Chennell Robin J. W. Willows Sean C. Warren David Carling Paul M. W. French Chris Dunsby Alessandro Sardini |
| author_sort | George Chennell |
| collection | DOAJ |
| description | We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase (AMPK) FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR), for fluorescence lifetime imaging (FLIM) readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP), in the original FRET biosensor, AMPK activity reporter (AMPKAR), with mTurquoise2 (mTq2FP), increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation. |
| first_indexed | 2024-04-14T05:22:31Z |
| format | Article |
| id | doaj.art-69f05b72ae6a49c8800bfdf46763383e |
| institution | Directory Open Access Journal |
| issn | 1424-8220 |
| language | English |
| last_indexed | 2024-04-14T05:22:31Z |
| publishDate | 2016-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Sensors |
| spelling | doaj.art-69f05b72ae6a49c8800bfdf46763383e2022-12-22T02:10:08ZengMDPI AGSensors1424-82202016-08-01168131210.3390/s16081312s16081312Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIMGeorge Chennell0Robin J. W. Willows1Sean C. Warren2David Carling3Paul M. W. French4Chris Dunsby5Alessandro Sardini6Cellular Stress Group, MRC Clinical Sciences Centre (CSC), Du Cane Road, London W12 0NN, UKCellular Stress Group, MRC Clinical Sciences Centre (CSC), Du Cane Road, London W12 0NN, UKThe Kinghorn Cancer Centre, Garvan Institute of Medical Research and St Vincent’s Clinical School, University of New South Wales, Darlinghurst, NSW 2010, AustraliaCellular Stress Group, MRC Clinical Sciences Centre (CSC), Du Cane Road, London W12 0NN, UKPhotonics Group, Department of Physics, Imperial College London, London SW7 2AZ, UKPhotonics Group, Department of Physics, Imperial College London, London SW7 2AZ, UKInstitute of Clinical Sciences (ICS), Department of Medicine, Imperial College London, Du Cane Road, London W12 0NN, UKWe describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase (AMPK) FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR), for fluorescence lifetime imaging (FLIM) readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP), in the original FRET biosensor, AMPK activity reporter (AMPKAR), with mTurquoise2 (mTq2FP), increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation.http://www.mdpi.com/1424-8220/16/8/1312FRETFLIMAMPKspheroid2-photonbiosensorTCSPC3D culture |
| spellingShingle | George Chennell Robin J. W. Willows Sean C. Warren David Carling Paul M. W. French Chris Dunsby Alessandro Sardini Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM Sensors FRET FLIM AMPK spheroid 2-photon biosensor TCSPC 3D culture |
| title | Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM |
| title_full | Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM |
| title_fullStr | Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM |
| title_full_unstemmed | Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM |
| title_short | Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM |
| title_sort | imaging of metabolic status in 3d cultures with an improved ampk fret biosensor for flim |
| topic | FRET FLIM AMPK spheroid 2-photon biosensor TCSPC 3D culture |
| url | http://www.mdpi.com/1424-8220/16/8/1312 |
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