Autoclaved methylcellulose

3D tumor spheroids were established by the hanging-drop method in a house-made spheroid media. The spheroid media was prepared by dissolving autoclaved methylcellulose (Sigma-Aldrich) at 2.4% (w/v) in preheated (60 °C), serum-free DMEM followed by dilution with an equivolume of DMEM supplemented with 10% FBS and an overnight stirring at 4 °C. The solution was then centrifuged at 5,000 xg for 2 hours at 4 °C to precipitate undissolved methylcellulose and the supernatant stored at 4 °C until use. To form each spheroid, we cultured 5,000 cells, either F98 glioma cells or F98 cells engineered to constitutively express a red fluorescent protein, mKate (F98-mKate; provided by Dr. Surojit Sur; Johns Hopkins University), in 20 μL spheroid media on the lid of a petri-dish. Spheroids were then grown upside down by covering a PBS-filled/moisturized petri-dish with the cell-seeded lid for 2 days. Finally, spheroids were transferred onto a sterile U-bottom 96-well plate (CELLSTAR®; Sigma-Aldrich) along with DMEM and incubated at 37⁰ C for 24 hours prior to their use for subsequent experiments.