Glioma and Astrocyte Cell Cultures Protocol

Human glioma LN18, LN229, U87-MG and U251 cell lines were from American Type Culture Collection (ATCC, Manassas, VA, USA); GBM patient-derived glioma primary cultures WG4 and IPIN20160420 were generated and cultured as previously described [20 (link)] in DMEM/Nutrient Mixture F-12, GlutaMAX™ medium, supplemented with 10% FBS (Gibco Life Technologies, Rockville, MD, USA) and antibiotics. Normal human astrocytes (NHA, Lonza Walkersville, MD, USA) were cultured in a commercial medium as described [33 (link)]. Temozolomide (TMZ, Sigma-Aldrich, Munich, Germany) was dissolved in water. Olaparib (OLA, MedChemExpress, Monmouth Junction, NJ, USA) was dissolved in DMSO.
For sphere cultures, LN18 glioma cells were seeded at a low density (1500 cells/cm²) on non-adherent plates and cultured in DMEM/F-12 GlutaMAX™ supplemented with 2% B27, 20 ng/mL rhuman bFGF, 20 ng/mL rhuman EGF, 0.0002% heparin and antibiotics (for details see Supplementary Table S2). Cells were fed every 3 days by adding 1 mL of the fresh medium.