title: Quantitative 3D cell-based assay performed with cellular spheroids and fluorescence microscopy.
abstract: Cell-based assays are essential in both basic research and drug discovery. Three-dimensional cellular spheroids are more realistic models of tumors and healthy tissues compared to standard two-dimensional cultures. Employing spheroids improves the reliability and the physiological significance of cell-based assays. We present a detailed drug assay protocol performed with live cellular spheroids. We employ automated epifluorescence live microscopy to investigate the effects of drugs on the spheroids over several days. We describe the spheroid preparation, manipulation, live fluorescence imaging, and data processing. We quantify the autophagy-triggering effects of the drugs (-)-Gossypol and Rapamycin in glioma cell spheroids. The formation of the autophagosomes and the fusion of the autophagosomes with lysosomes in the treated spheroids are monitored over time and space with a mRFP:GFP:LC3 fusion protein.
authors: Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt, Frankfurt am Main, Germany.
authors: Nariman
authors: N
authors: Ansari
authors: Stefanie
authors: S
authors: Müller
authors: Ernst H K
authors: EH
authors: Stelzer
authors: Francesco
authors: F
authors: Pampaloni
mesh_terms: D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003470:Culture Media; D004353:Drug Evaluation, Preclinical / Q000379:methods; D006072:Gossypol / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D059010:Single-Cell Analysis / Q000379:methods*; D020123:Sirolimus / Q000494:pharmacology; D018874:Spheroids, Cellular / Q000166:cytology*
chemical_list: D003470:Culture Media; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006072:Gossypol; D020123:Sirolimus