Glioma Cell Culture and Hypoxia Conditions

Human D54MG, U373MG and SNB191 glioma cell lines and primary human GBM XD45 and JX10 xenolines (UAB Brain Tumor Specialized Program Of Research Excellence) were cultured in Dulbecco’s modified Eagle’s medium (Gibco-BRL, Paisley, UK) supplemented with 10% heat-inactivated fetal bovine serum, L-glutamine, penicillin/streptomycin and non-essential amino acids (all Gibco-BRL) (27 (link),28 (link)). The cells were cultured at 37°C, in a humidified atmosphere of 5% CO₂ and 95% air (~21% pO₂). For the hypoxia experiments, the cells were kept for the indicated durations in a cell culture incubator (I-Glove; BioSpherix, Inc., Lacona, NY, USA) with an oxygen level set to 1 or 5% pO₂, as indicated. Chloroquine was purchased from Sigma-Aldrich (St. Louis, MO, USA) and the wide-spectrum MMP-inhibitor, GM6001, was obtained from Enzo Life Sciences Inc., (Farmingdale, NY, USA).