96 well flat clear bottom white polystyrene tc treated microplates

Spheroids were formed in Corning® 96 Well Flat Clear Bottom White Polystyrene TC-Treated Microplates (Corning, MA, USA). Wells were coated in 1.5% UltraPure™ Agarose (Invitrogen Corporation, CA, USA) solution prepared in DMEM. GIST-T1 and GIST430 cells were suspended atop the agar layer in complete DMEM (9,000 cells/well) and left undisturbed for 96 hours at 37°C and 5% CO₂. Resulting spheroids were treated with appropriate drug(s) in 50 μl complete DMEM. Spheroids were imaged at 4x magnification by EVOS™ FL Digital Inverted Microscope (AMG, WA, USA) after 72 hours of drug treatment. Spheroid surface area was measured using ImageJ software (NIH, MD, USA). The CellTiter-Glo® Luminescent Cell Viability Assay (Promega, WI, USA) was performed after imaging, with luminescence measured by EnVision Plate Reader. Three independent experiments were performed with a minimum of three technical replicates in each treatment arm. Statistical analyses were conducted using GraphPad Prism Version 6.05 (GraphPad Software, CA, USA). Surface area and viability of treated spheroids were normalized to vehicle-treated spheroids of the same cell line. Comparison of treatment arms was performed with one-way ANOVA. Post-hoc comparisons were made using the Bonferroni multiple comparisons method.