Monitoring of apoptosis in 3D cell cultures by FRET and light sheet fluorescence microscopy

International Journal of Molecular Sciences
Petra WeberHerbert Schneckenburger

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.

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Citations

Apr 1, 2017·Optics Letters·Claire A MitchellSimon M Ameer-Beg
Jul 10, 2019·Biotechnology and Bioengineering·Elisabete C CostaIlídio J Correia
Sep 6, 2019·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Irina A OkkelmanRuslan I Dmitriev
Nov 13, 2019·Methods and Applications in Fluorescence·Herbert Schneckenburger
Feb 7, 2020·Methods and Applications in Fluorescence·Chetan PoudelClemens F Kaminski
Feb 6, 2017·The Review of Scientific Instruments·H SparksP M W French
Jun 6, 2020·Biomedical Optics Express·Peter F FavreauMelissa C Skala
May 8, 2021·Journal of Cell Science·Ruslan I DmitrievMargarida M Barroso
Sep 22, 2021·ACS Nano·Adam J Bowman, Mark A Kasevich

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Methods Mentioned

BETA
Fluorescence
Fluorescence Microscopy
FRET
PMA
elastic light scattering
ELISA

Related Concepts

Related Feeds

Apoptosis

Apoptosis is a specific process that leads to programmed cell death through the activation of an evolutionary conserved intracellular pathway leading to pathognomic cellular changes distinct from cellular necrosis

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