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.
Physical Biology Group, Buchmann Institute for Molecular Life Sciences (BMLS), Goethe Universität Frankfurt am Main, Max-von-Laue-Str, 15D-60438, Frankfurt am Main, Germany.
Nariman
N
Ansari
Stefanie
S
Hardung
Katharina
K
Hötte
Stefanie
S
Rakel
Patrick
P
Antonietti
Donat
D
Kögel
Ernst H K
EH
Stelzer
Francesco
F
Pampaloni
D001343:Autophagy / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D006072:Gossypol / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D020123:Sirolimus / Q000494:pharmacology; D018874:Spheroids, Cellular / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D000097573:Red Fluorescent Protein
D006072:Gossypol; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D020123:Sirolimus; D000097573:Red Fluorescent Protein