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T98G, U87MG, TS603 and 13.0302 cells were cultured in neurobasal medium (Stemcell technologies, 5751) containing 10% proliferation supplement (Stemcell technologies, 5751), 20ng/mL H-EGF (Stemcell technologies, 78006), 10ng/mL H-bFGF (Stemcell technologies, 78003) and 2μg/mL Heparin (Stemcell technologies, 7980). Once neurospheres were formed, they were dissociated by Accutase (VWR, 490007-741) into single cells. Single cells were seeded into 12-well plates (5,000 cells/well) and treated with control medium or THIO at 5 μM for 12 days. Phase contrast images of neurospheres were acquired by ECHO Revolve Microscope at day 3, 6, 9 and 12. At day 12, neurospheres were stained using Live Dead Cell Viability Assay Kit (Sigma-Aldrich, CBA415) and imaged by ECHO Revolve Microscope.

T98G, U87MG, TS-603, and 13.0302 cells were cultured in neurobasal medium (Stemcell Technologies, 5751) containing 10% proliferation supplement (Stemcell Technologies, 5751), 20 ng/mL H-EGF (Stemcell Technologies, 78006), 10 ng/mL H-bFGF (Stemcell Technologies, 78003), and 2 μg/mL Heparin (Stemcell Technologies, 7980). Once neurospheres were formed, they were dissociated by Accutase (VWR, 490007–741) into single cells. Single cells were seeded into 12-well plates (5,000 cells/well) and treated with control medium or Gamitrinib at 1, 2.5, and 5 μmol/L for 12 days. Phase contrast images of neurospheres were acquired by ECHO Revolve Microscope at days 3, 6, 9, and 12. At day 12, neurospheres were stained using Live Dead Cell Viability Assay Kit (Sigma-Aldrich, CBA415) and imaged by ECHO Revolve Microscope. Two independent experiments were performed.