The study was approved by The Ethics Board at the Children’s Memorial Health Institute, Warsaw, Poland. The samples of patients’ SEGAs were analyzed after written consent was obtained from their parents. Patients were diagnosed as having TSC according to Roach’s criteria. The patients presented with acute hydrocephalus and were operated after large SEGAs were revealed in brain MRI (Additional file 3 : Figure S3a). Freshly resected SEGA samples from two patients were cut into small pieces and trypsynized for 1 h at 37 °C. After trypsinization the tissue fragments were dispersed with a pipette to small clumps or single cells. The obtained cell suspension was centrifuged and the pellet was suspended in DMEM 4.5 g/l glucose supplemented with 5 % fetal bovine serum (FBS; Gibco, Karlsruhe, Germany) and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin; Sigma, St. Louis, MO). Cells were maintained for about 2 weeks until they reached confluence and were used for experiments.
For live imaging experiments, cells were plated on gelatin-coated μ-Slide VI 0.4 plates (Ibidi, Planegg, Germany). During pharmacological treatment, the medium was changed every second day and the drugs were used as follows: U0126 (20 μM), rapamycin (20 nM), L-BSO (20 or 100 μM).
For live imaging experiments, cells were plated on gelatin-coated μ-Slide VI 0.4 plates (Ibidi, Planegg, Germany). During pharmacological treatment, the medium was changed every second day and the drugs were used as follows: U0126 (20 μM), rapamycin (20 nM), L-BSO (20 or 100 μM).