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.
Cells 2018, 7(6), 57; https://doi.org/10.3390/cells7060057
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)
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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Keywords:
apoptosis; breast cancer; caspase-3; fluorescence lifetime imaging; Förster resonance energy transfer
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MDPI and ACS Style
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. https://doi.org/10.3390/cells7060057
AMA Style
Buschhaus JM, Humphries B, Luker KE, Luker GD. A Caspase-3 Reporter for Fluorescence Lifetime Imaging of Single-Cell Apoptosis. Cells. 2018; 7(6):57. https://doi.org/10.3390/cells7060057
Chicago/Turabian StyleBuschhaus, Johanna M.; Humphries, Brock; Luker, Kathryn E.; Luker, Gary D. 2018. "A Caspase-3 Reporter for Fluorescence Lifetime Imaging of Single-Cell Apoptosis" Cells 7, no. 6: 57. https://doi.org/10.3390/cells7060057
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