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.
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References
Levine B, Kroemer G (2008) Autophagy in the pathogenesis of disease. Cell 132:27–42
Levine B (2007) Cell biology: autophagy and cancer. Nature 446:745–747
Klionsky DJ, Abdalla FC, Abeliovich H et al (2012) Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8:445–544
Pampaloni F, Reynaud EG, Stelzer EH (2007) The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol 8:839–845
Friedrich J, Seidel C, Ebner R, Kunz-Schughart LA (2009) Spheroid-based drug screen: considerations and practical approach. Nat Protoc 4:309–324
Pampaloni F, Ansari N, Stelzer EH (2013) High-resolution deep imaging of live cellular spheroids with light-sheet-based fluorescence microscopy. Cell Tissue Res 352:161–177
Pampaloni F, Stelzer EH, Masotti A (2009) Three-dimensional tissue models for drug discovery and toxicology. Recent Pat Biotechnol 3:103–117
Voss V, Senft C, Lang V, Ronellenfitsch MW, Steinbach JP, Seifert V, Kogel D (2010) The pan-Bcl-2 inhibitor (-)-gossypol triggers autophagic cell death in malignant glioma. Mol Cancer Res 8:1002–1016
Kimura S, Noda T, Yoshimori T (2007) Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy 3:452–460
Hundeshagen P, Hamacher-Brady A, Eils R, Brady NR (2011) Concurrent detection of autolysosome formation and lysosomal degradation by flow cytometry in a high-content screen for inducers of autophagy. BMC Biol 9:38
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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Ansari, N. et al. (2014). Quantifying the Autophagy-Triggering Effects of Drugs in Cell Spheroids with Live Fluorescence Microscopy. In: Robles-Flores, M. (eds) Cancer Cell Signaling. Methods in Molecular Biology, vol 1165. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0856-1_3
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DOI: https://doi.org/10.1007/978-1-4939-0856-1_3
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0855-4
Online ISBN: 978-1-4939-0856-1
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