Glioblastoma Cell Culturing and TMZ Treatment

Human GBM T98G (ATCC, CRL-1690) and U87 cells (ATCC, CRL-1690) were routinely cultivated in high glucose (4500 g/L) DMEM medium (Sigma, St. Louis, MO, USA), which was supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% Antibiotic-Antimycotic Solution (Sigma) [59 (link),60 (link)]. For endpoint experiments, the cells were harvested with 0.25% trypsin in Ca²⁺/Mg²⁺-free PBS (Corning, Corning, NY, USA) supplemented with 0.5 mM EDTA (UltraPure^TM; Invitrogen, Carlsbad, CA, USA), counted with Z2 particle counter (Beckman-Coulter, Brea, CA, USA) and then seeded into multi-well tissue culture plates (Falcon). For some experiments U373 (human astrocytoma; ATCC, HTB-17), U118 (ATCC, HTB-15), Ln229 (ATCC, CRL-2611), Ln18 (ATCC, CRL-2610) and F98 cells (rat glioblastoma; ATCC, CRL-2397) were cultivated, as described above. TMZ (Sigma; No. T2577) was dissolved in DMSO at the concentration of 100 mM, stored in −80 °C, and administered at the concentrations of 5 or 25 μM. Unless stated otherwise, the medium containing 0.25‰ DMSO (corresponding to 25 μM TMZ) was used as a vesicle control.