Cargando…
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...
Autores principales: | , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2016
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5017477/ https://www.ncbi.nlm.nih.gov/pubmed/27548185 http://dx.doi.org/10.3390/s16081312 |
_version_ | 1782452757085552640 |
---|---|
author | Chennell, George Willows, Robin J. W. Warren, Sean C. Carling, David French, Paul M. W. Dunsby, Chris Sardini, Alessandro |
author_facet | Chennell, George Willows, Robin J. W. Warren, Sean C. Carling, David French, Paul M. W. Dunsby, Chris Sardini, Alessandro |
author_sort | Chennell, George |
collection | PubMed |
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. |
format | Online Article Text |
id | pubmed-5017477 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-50174772016-09-22 Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM Chennell, George Willows, Robin J. W. Warren, Sean C. Carling, David French, Paul M. W. Dunsby, Chris Sardini, Alessandro Sensors (Basel) Article 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. MDPI 2016-08-19 /pmc/articles/PMC5017477/ /pubmed/27548185 http://dx.doi.org/10.3390/s16081312 Text en © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Chennell, George Willows, Robin J. W. Warren, Sean C. Carling, David French, Paul M. W. Dunsby, Chris Sardini, Alessandro Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM |
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 | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5017477/ https://www.ncbi.nlm.nih.gov/pubmed/27548185 http://dx.doi.org/10.3390/s16081312 |
work_keys_str_mv | AT chennellgeorge imagingofmetabolicstatusin3dcultureswithanimprovedampkfretbiosensorforflim AT willowsrobinjw imagingofmetabolicstatusin3dcultureswithanimprovedampkfretbiosensorforflim AT warrenseanc imagingofmetabolicstatusin3dcultureswithanimprovedampkfretbiosensorforflim AT carlingdavid imagingofmetabolicstatusin3dcultureswithanimprovedampkfretbiosensorforflim AT frenchpaulmw imagingofmetabolicstatusin3dcultureswithanimprovedampkfretbiosensorforflim AT dunsbychris imagingofmetabolicstatusin3dcultureswithanimprovedampkfretbiosensorforflim AT sardinialessandro imagingofmetabolicstatusin3dcultureswithanimprovedampkfretbiosensorforflim |