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.