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Int. J. Mol. Sci. 2015, 16(3), 5375-5385; doi:10.3390/ijms16035375

Monitoring of Apoptosis in 3D Cell Cultures by FRET and Light Sheet Fluorescence Microscopy

1
Institute of Applied Research, Aalen University, Anton-Huber Str. 21, 73430 Aalen, Germany
2
Cellendes GmbH, Markwiesenstr. 55, 72770 Reutlingen, Germany
*
Author to whom correspondence should be addressed.
Academic Editor: Anthony Lemarié
Received: 16 January 2015 / Revised: 12 February 2015 / Accepted: 26 February 2015 / Published: 9 March 2015
(This article belongs to the Special Issue Förster Resonance Energy Transfer (FRET) 2015)
View Full-Text   |   Download PDF [1544 KB, uploaded 9 March 2015]   |  

Abstract

Non-radiative cell membrane associated Förster Resonance Energy Transfer (FRET) from an enhanced cyan fluorescent protein (ECFP) to an enhanced yellow fluorescent protein (EYFP) is used for detection of apoptosis in 3-dimensional cell cultures. FRET is visualized in multi-cellular tumor spheroids by light sheet based fluorescence microscopy in combination with microspectral analysis and fluorescence lifetime imaging (FLIM). Upon application of staurosporine and to some extent after treatment with phorbol-12-myristate-13-acetate (PMA), a specific activator of protein kinase c, the caspase-3 sensitive peptide linker DEVD is cleaved. This results in a reduction of acceptor (EYFP) fluorescence as well as a prolongation of the fluorescence lifetime of the donor (ECFP). Fluorescence spectra and lifetimes may, therefore, be used for monitoring of apoptosis in a realistic 3-dimensional system, while light sheet based microscopy appears appropriate for 3D imaging at low light exposure. View Full-Text
Keywords: energy transfer; FRET; apoptosis; light sheet fluorescence microscopy (LSFM); single plane fluorescence microscopy (SPIM); fluorescence lifetime imaging (FLIM); microspectral analysis; cell spheroids energy transfer; FRET; apoptosis; light sheet fluorescence microscopy (LSFM); single plane fluorescence microscopy (SPIM); fluorescence lifetime imaging (FLIM); microspectral analysis; cell spheroids
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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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MDPI and ACS Style

Weber, P.; Schickinger, S.; Wagner, M.; Angres, B.; Bruns, T.; Schneckenburger, H. Monitoring of Apoptosis in 3D Cell Cultures by FRET and Light Sheet Fluorescence Microscopy. Int. J. Mol. Sci. 2015, 16, 5375-5385.

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