Dmem ham f10 1 1 medium

The astrocytoma cell line U373 was obtained from the American Type Culture Collection (Rockville, MD, USA); cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/HAM F10 (1:1) supplemented with 50 units/ml penicillin, 50 μg/ml streptomycin, and 10 % FCS on poly-l-lysine-coated plates.
For astrocytes-enriched human cell cultures, fetal brain tissue (15–20 weeks of gestation) was obtained from spontaneous or medically induced abortions with appropriate maternal written consent for brain autopsy. Tissue was obtained in accordance with the Declaration of Helsinki and the AMC Research Code provided by the Medical Ethics Committee of the AMC. Resected tissue samples were collected in DMEM/HAM F10 (1:1) medium (Gibco, Life Technologies), supplemented with 50 units/ml penicillin and 50 μg/ml streptomycin and 10 % fetal calf serum (FCS). Cell isolation was performed as previously described [25 (link)].

Primary fetal astrocyte-enriched cell cultures were derived from human fetal brain tissue (14–20 weeks of gestation) obtained from medically induced abortions. All material was collected from donors from whom a written informed consent for the use of the material for research purposes was obtained by the Bloemenhove clinic. Tissue was obtained in accordance with the Declaration of Helsinki and the Amsterdam UMC Research Code provided by the Medical Ethics Committee. Tissue samples were collected in DMEM/HAM F10 (1:1) medium (Gibco/ThermoFisher Scientific, Waltham, MA, USA), supplemented with 1% penicillin/streptomycin and 10% fetal calf serum (FCS). Primary cell cultures of astrocytes were prepared as previously described [49 (link)]. The culture medium was subsequently refreshed twice a week. Cultures reached confluence after 2–3 weeks. Astrocytes were used for analyses at passages 2–5. More than 98% of the cells in primary culture, as well as in the successive 12 passages were strongly immunoreactive for the astrocytic marker glial fibrillary acid protein (GFAP) and S100β as previously reported [45 (link)].

Primary TSC cell cultures were derived from surgical brain specimens obtained from two patients (age at surgery: 2.5 and 2 years; gender: male; mutation: TSC2) undergoing epilepsy surgery at the Wilhelmina Children's Hospital of the University Medical Center Utrecht (Utrecht, The Netherlands). Cell isolation was performed as described elsewhere 38, 39. Briefly, after removal of blood vessels, tissue was mechanically minced into smaller fragments and enzymatically digested by incubating at 37°C for 30 min with 2.5% trypsin (Sigma‐Aldrich). Tissue was washed with incubation medium containing Dulbecco's modified Eagle's medium (DMEM)/HAM F10 (1:1) medium (Gibco, Life Technologies, Grand Island, NY, USA), supplemented with 1% penicillin/streptomycin and 10% foetal calf serum (Gibco, Life Technologies) and triturated by passing through a 70‐μm mesh filter. The cell suspension was incubated at 37°C, 5% CO₂ for 48 h to let cells adhere the culture flask before it was washed with PBS to remove excess of myelin and cell debris. Cultures were subsequently refreshed twice a week.