Glomax explorer reader

Cells were seeded (2 × 10³ or 5 × 10³ cells per well) into a 96-well plate with 10% FBS 1% PSG for 24 h before the treatments were applied. Cells were then treated with 0, 1, 5 or 10 µM of the stated KIs. Following 24 h (GBM cell lines) or 48 h (GSCs) treatment, cells were fixed with 4% paraformaldehyde (PFA) in 1x phosphate buffered saline (PBS) (Sigma-Aldrich) and incubated at room temperature (temp) for 30 min. PFA was removed and plates left to dry at room temp for 10 min. They were then washed once with PBS and stained with 0.1% crystal violet solution at room temp for 30 min. The solution was then aspirated and washed in 1x PBS twice and water once and left open overnight to dry. When ready to read, 50 μl of 10% acetic acid was added to each well, plates were agitated on a plate rocker for 20 min and absorbance was recorded at 600 nm using a GloMax Explorer reader (Promega). Graphs show the mean ± SD of at least three independent experiments.

Cells were seeded (2 × 10³ cells per well) into a 96-well plate with 10% FBS 1% PSG for 24 h before the treatments were applied. Cells were then treated with DMSO or 1 µM of the stated KIs. Following 24 h (GBM cell lines) or 48 h (GSCs) treatment, 10 μl of water-soluble tetrazolium salts-1 (WST-1) reagent was added to each well and incubated at 37 °C, 5% CO₂ for 2 h. The plate was then agitated on a plate rocker at room temp for 1 min and the absorbance recorded at 450 nm using a GloMax Explorer reader (Promega). Graphs show the mean ± SD of at least three independent experiments.

Following the cell invasion assay, cell viability was assessed using CellTiter-Glo luminescent cell viability assay (Promega). Cells were incubated at room temp for 30 min before 100 µl CellTiter-Glo reagent was added to each well, agitated on a plate rocker for 2 min and incubated at room temp for 10 min. Luminescence was recorded with an integration time of 0.3 s using a GloMax Explorer reader (Promega). Graphs show the mean ± SD of at least three independent experiments.