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Quantifying the Autophagy-Triggering Effects of Drugs in Cell Spheroids with Live Fluorescence Microscopy

  • Nariman Ansari
  • Stefanie Hardung
  • Katharina Hötte
  • Stefanie Rakel
  • Patrick Antonietti
  • Donat Kögel
  • Ernst H. K. Stelzer
  • Francesco PampaloniEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1165)

Abstract

We present a 3D assay for the quantification of the autophagic flux in live cell spheroids by using the fluorescent reporter mRFP-GFP-LC3. The protocol describes the formation of the spheroids from the astrocytoma cell line U343, live long-term 3D fluorescence imaging of drug-treated spheroids, and the image processing workflow required to extract quantitative data on the autophagic flux.

Key words

Three-dimensional cell cultures Live-cell assay Glioma tumor spheroids Autophagic flux Drug development Live cell imaging Automated fluorescence microscopy Live confocal microscopy 

Notes

Acknowledgements

The authors thank the German Federal Ministry of Education and Research (BMBF) for financial support (project ProMEBS) and Dr. Tamotsu Yoshimori (National Institute of Genetics, Shizuoka, Japan) for providing the mRFP-GFP-LC3 plasmid.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Nariman Ansari
    • 1
  • Stefanie Hardung
    • 1
  • Katharina Hötte
    • 1
  • Stefanie Rakel
    • 2
  • Patrick Antonietti
    • 2
  • Donat Kögel
    • 2
  • Ernst H. K. Stelzer
    • 1
  • Francesco Pampaloni
    • 1
    Email author
  1. 1.Physical Biology GroupBuchmann Institute for Molecular Life Sciences (BMLS), Goethe Universität Frankfurt am MainFrankfurt am MainGermany
  2. 2.Experimental NeurosurgeryNeuroscience Center, Goethe University HospitalFrankfurt am MainGermany

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