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Cells 2018, 7(6), 57; https://doi.org/10.3390/cells7060057

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

1
Center for Molecular Imaging, Department of Radiology, University of Michigan, Ann Arbor, MI 48190, USA
2
Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48190, USA
3
Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48190, USA
*
Author to whom correspondence should be addressed.
Received: 25 April 2018 / Revised: 1 June 2018 / Accepted: 8 June 2018 / Published: 13 June 2018
(This article belongs to the Special Issue Innovative Methods to Monitor Single Live Cells)
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Abstract

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. View Full-Text
Keywords: apoptosis; breast cancer; caspase-3; fluorescence lifetime imaging; Förster resonance energy transfer apoptosis; breast cancer; caspase-3; fluorescence lifetime imaging; Förster resonance energy transfer
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).
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Buschhaus, J.M.; Humphries, B.; Luker, K.E.; Luker, G.D. A Caspase-3 Reporter for Fluorescence Lifetime Imaging of Single-Cell Apoptosis. Cells 2018, 7, 57.

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