Cultivation of Glioblastoma and Neuroglioma Cell Lines

U87 (brain-like glioblastoma), A172 (glioblastoma), and H4 (neuroglioma) cell lines were generously supplied by Professor Carla Vitorino (Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal). U118 (astrocytoma/glioblastoma) and U373 (astrocytoma/glioblastoma) cell lines were kindly gifted by Professor Maria Conceição Pedroso de Lima (Faculty of Sciences and Technology, Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal). HEK-293 (immortalized human embryonic kidney fibroblasts) was kindly provided by Professor Paulo Oliveira (Institute for Interdisciplinary Research, Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal). Cells were cultivated in Dulbecco’s Modified Eagle’s Medium-high glucose (DMEM-HG) (Biowest, Nuaille’, France) supplemented with 10% (v/v) of heat-inactivated fetal bovine serum (FBS) (Sigma, St. Louis, MO, USA), and 1% (v/v) of penicillin-streptomycin (Sigma, St. Louis, MO, USA). Cells were maintained in a humidified atmosphere at 37 °C and 5% CO₂. Throughout the experiments, cells were used at 80% confluence, and monitored by microscope observation to detect any morphological changes.