Human pediatric cerebellar glioblastoma multiforme GBM6840 cells were cultured in DMEM (high glucose) supplemented with 10 % FBS. Cell viability studies and IC 50 determinations were conducted by live/dead cell staining and flow cytometric analysis. Briefly, cells were seeded in 96 well-plates ( 4 cells/well). The next day, ATV (solubilized in DMSO or formulated with A-pBuOzi-A) was added to the medium at a final concentration of 0 to 80 µM. The corresponding concentrations of DMSO and A-pBuOzi-A alone served as controls. After 48 h incubation, cells were rinsed with PBS, trypsinized, and resuspended in 250 µL PBS containing 0.5 g/L soybean trypsin inhibitor (Invitrogen, Carlsbad, USA). Cell culture medium, wash solution and trypsinized cells were collected and incubated for 15 min at 22 °C with the live cell indicator calcein-AM (Invitrogen, Carlsbad, USA) at a final concentration of 10 nM. Immediately before flow cytometry, cell suspensions were incubated with the dead cell indicator propidium iodide (1 mg/L, Invitrogen) for 1 min. Cells were analyzed on a FACS Calibur flow cytometer (Beckton Dickinson, Heidelberg, Germany); at least 5,000 cells were analyzed per condition. Data are presented as means ± SEM of n=3 independent experiments. Cell viability was expressed as the percentage of calcein-positive cells. IC 50 values were determined by non-linear regression (log(inhibitor) vs. response -variable slope (four parameters)) using Prism v7.04 (GraphPad, San Diego, U.S.A.).