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A Caspase-3 Reporter for Fluorescence Lifetime Imaging of Single-Cell Apoptosis

Fluorescence lifetime imaging (FLIM) is a powerful imaging modality used to gather fluorescent reporter data independent of intracellular reporter intensity or imaging depth. We applied this technique to image real-time activation of a reporter for the proteolytic enzyme, caspase-3, in response to a...

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Detalles Bibliográficos
Autores principales: Buschhaus, Johanna M., Humphries, Brock, Luker, Kathryn E., Luker, Gary D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6025355/
https://www.ncbi.nlm.nih.gov/pubmed/30720785
http://dx.doi.org/10.3390/cells7060057
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author Buschhaus, Johanna M.
Humphries, Brock
Luker, Kathryn E.
Luker, Gary D.
author_facet Buschhaus, Johanna M.
Humphries, Brock
Luker, Kathryn E.
Luker, Gary D.
author_sort Buschhaus, Johanna M.
collection PubMed
description Fluorescence lifetime imaging (FLIM) is a powerful imaging modality used to gather fluorescent reporter data independent of intracellular reporter intensity or imaging depth. We applied this technique to image real-time activation of a reporter for the proteolytic enzyme, caspase-3, in response to apoptotic cell death. This caspase-3 reporter activity provides valuable insight into cancer cell responsiveness to therapy and overall viability at a single-cell scale. Cleavage of a aspartate-glutamate-valine-aspartate (DEVD) linkage sequence alters Förster resonance energy transfer (FRET) within the reporter, affecting its lifetime. Cellular apoptosis was quantified in multiple environments ranging from 2D flat and 3D spheroid cell culture systems to in vivo murine mammary tumor xenografts. We evaluated cell-by-cell apoptotic responses to multiple pharmacological and genetic methods of interest involved in cancer cell death. Within this article, we describe methods for measuring caspase-3 activation at single-cell resolution in various complex environments using FLIM.
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spelling pubmed-60253552018-07-09 A Caspase-3 Reporter for Fluorescence Lifetime Imaging of Single-Cell Apoptosis Buschhaus, Johanna M. Humphries, Brock Luker, Kathryn E. Luker, Gary D. Cells Protocol Fluorescence lifetime imaging (FLIM) is a powerful imaging modality used to gather fluorescent reporter data independent of intracellular reporter intensity or imaging depth. We applied this technique to image real-time activation of a reporter for the proteolytic enzyme, caspase-3, in response to apoptotic cell death. This caspase-3 reporter activity provides valuable insight into cancer cell responsiveness to therapy and overall viability at a single-cell scale. Cleavage of a aspartate-glutamate-valine-aspartate (DEVD) linkage sequence alters Förster resonance energy transfer (FRET) within the reporter, affecting its lifetime. Cellular apoptosis was quantified in multiple environments ranging from 2D flat and 3D spheroid cell culture systems to in vivo murine mammary tumor xenografts. We evaluated cell-by-cell apoptotic responses to multiple pharmacological and genetic methods of interest involved in cancer cell death. Within this article, we describe methods for measuring caspase-3 activation at single-cell resolution in various complex environments using FLIM. MDPI 2018-06-13 /pmc/articles/PMC6025355/ /pubmed/30720785 http://dx.doi.org/10.3390/cells7060057 Text en © 2018 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 Protocol
Buschhaus, Johanna M.
Humphries, Brock
Luker, Kathryn E.
Luker, Gary D.
A Caspase-3 Reporter for Fluorescence Lifetime Imaging of Single-Cell Apoptosis
title A Caspase-3 Reporter for Fluorescence Lifetime Imaging of Single-Cell Apoptosis
title_full A Caspase-3 Reporter for Fluorescence Lifetime Imaging of Single-Cell Apoptosis
title_fullStr A Caspase-3 Reporter for Fluorescence Lifetime Imaging of Single-Cell Apoptosis
title_full_unstemmed A Caspase-3 Reporter for Fluorescence Lifetime Imaging of Single-Cell Apoptosis
title_short A Caspase-3 Reporter for Fluorescence Lifetime Imaging of Single-Cell Apoptosis
title_sort caspase-3 reporter for fluorescence lifetime imaging of single-cell apoptosis
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6025355/
https://www.ncbi.nlm.nih.gov/pubmed/30720785
http://dx.doi.org/10.3390/cells7060057
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