Quantifying the Autophagy-Triggering Effects of Drugs in Cell Spheroids with Live Fluorescence Microscopy
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 wordsThree-dimensional cell cultures Live-cell assay Glioma tumor spheroids Autophagic flux Drug development Live cell imaging Automated fluorescence microscopy Live confocal microscopy
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.