U87mg

The human glioma cell line U87MG was obtained from LGC Standards (Wesel, Germany) and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin (all from Sigma-Aldrich, Taufkirchen, Germany). Six to eight week old male NMRI nude mice were injected stereotactically with 50.000 U87MG-tdTomato cells in 2 μl PBS. Implantation was performed into the right hemisphere, 1 mm rostral and 2 mm lateral from the Bregma at a depth of 500 μm (n = 5 mice, Charles River, Sulzfeld, Germany) using a Hamilton syringe, driven by a fine step motor. Animals were anesthetized with ketamine/xylazine and unresponsive to stimuli during the intracranial injection. MRI was performed on day 16 and 28 post tumor cell implantation.

The human GB cell line U87MG was obtained from the ATCC (LGC Promochem, Molsheim, France) and expanded in DMEM-high glucose medium (DMEM-HG, Ozyme) containing 10% FBS (Fisher Scientific, Illkirch, France) and 1% antibiotics (Sigma-Aldrich). Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (Verviers, Belgium). Cells were cultured, according to the supplier’s instructions, in endothelial cell growth medium-2 (EGM-2), corresponding to endothelial basal medium-2 (EBM-2) containing the supplements and growth factors of the EGM-2 SingleQuot™ kit (Lonza). U87MG cells and HUVECs were maintained under an atmosphere containing 5% CO₂ (37 °C), in a humidified incubator, until they reached 80% confluence.

We chose two human brain cell lines for our studies: The human neuroblastoma cell line SH-SY5Y (Cat.No. CRL-2266, ATCC LGC Standards GmbH, Wesel, Germany) and the human primary glioblastoma cell line U-87 MG (Cat.No. 300367, CLS Cell Lines Service GmbH, Eppelheim, Germany).
U-87 MG and SH-SY5Y cells were cultured in 1:1 MEM Eagle/Ham’s F12 medium containing Earle’s salts, L-glutamine, and sodium bicarbonate (Cat.No. M4655 and N6658, Sigma-Aldrich Chemie GmbH, Munich, Germany) supplemented with 10% fetal bovine serum (Cat.No. S0615, Biochrom GmbH, Berlin, Germany) and 1% penicillin/streptomycin (Cat.No. P0781, Sigma-Aldrich Chemie GmbH, Munich, Germany). Both cell lines were incubated in separate culture flasks at 37 °C in a 95% air and 5% CO₂ atmosphere. Cell culture medium was changed every 2–3 days. A mixture of phosphate-buffered saline (PBS, Cat.No. 18912014, Gibco Thermo Fisher Scientific, Waltham, MA, USA), 0.025% (w/v) trypsin, and 0.011% (w/v) ethylenediaminetetraacetic acid (EDTA, Cat.No. L2143, Biochrom GmbH, Berlin, Germany) was applied for 3–4 min to detach the cells prior to cell counting and seeding.