Ca2 mg2 free pbs

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.

Human glioblastoma multiforme T98G cells (ATCC, CRL-1690) were cultured in standard conditions with high levels of glucose (4500 g/L) DMEM medium (Sigma), supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% antibiotic-antimycotic Solution (Sigma) as described previously [43 (link)]. For each experiment, cells were harvested with Ca^2+/Mg²⁺-free PBS (Corning) with 0.5 mM EDTA (UltraPureTM; Invitrogen) solution, counted in a Z2 particle counter (Beckman Coulter), and seeded at an appropriate density into multi-well tissue culture plates (Eppendorf). Human epidermal growth factor (EGF; Sigma; No. E9644) was dissolved in 10 mM acetic acid/0.1% BSA and stored at −80 °C. EGFR inhibitor erlotinib (Erl; Sigma; No. SML2156) stock solution (4.6 mM; 9.2 µM in cell culture media) was prepared in DMSO (Sigma). N-Acetyl-L-cysteine (NAC; Sigma; No. A9165) was dissolved in sterile-filtered H₂O to 1 M and used in cell culture at 1 mM final concentration.