Multiwell tissue culture plates

Mice (C57BL6/J wildtype [A_2AR^+/+] and A_2AR-knockout [A_2AR^−/−]) were injected intraperitoneally with 2 ml of sterile Brewer's thioglycolate broth (4% w/v). Four days later, the mice were terminated by cervical dislocation, and peritoneal macrophage cells (IPMΦ) were harvested using 10 ml of Dulbecco's modified Eagle's medium (DMEM; catalog no.: LM-D1111; GENTAUR Europe BVBA). The cells were centrifuged for 10 min at 300g at 4 °C. The cells were suspended in 2 ml red blood cell lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃, and 0.1 mM EDTA), incubated for a few minutes on ice, and the volume was brought up to 10 ml with DMEM solution. Macrophages were centrifuged again for 10 min at 300g at 4 °C and resuspended in DMEM containing 10% fetal bovine serum (FBS), 50 U/ml penicillin, 50 μg/ml streptomycin, and 1.5 mg/ml sodium bicarbonate. Cells were seeded into Falcon Multiwell tissue culture plates. The dishes were then incubated at 37 °C in a humidified incubator for 5 h to allow the cells to adhere to the surface of the dishes. Nonadherent cells were removed by washing with serum-free DMEM, and the cells were suspended again in DMEM containing 10% FBS. About 18 h after the initial plating, the macrophages were treated with the various pharmacological compounds.

Cells were seeded at a density of 8.0 × 10⁵–1.2 × 10⁵ cells/cm² into either multiwell tissue culture plates (BD Falcon) or into Ibidi μ-slides, in the appropriate culture media. Cells were placed in the INCUCYTE^TM Live-Cell Imaging System (Essen Bioscience). Images were acquired every 5–30 minutes, depending on the experiment. Data was exported using INCUCYTE^TM software, provided by the manufacturer.

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.