Astrocyte growth supplement

LN18, LN229, and NCH82 glioblastoma cell lines were grown in cDMEM containing 10% FCS, 2 mM Glutamine, and 50 µg/ml Gentamycin. Cell lines were authenticated using Multiplex Cell Authentication by Multiplexion GmbH (Heidelberg, Germany). The SNP profiles matched known profiles or were unique. Primary human astrocytes (ScienCell, Carlsbad, CA, USA) were grown in AM medium containing 2% FCS, 1% astrocyte growth supplement (ScienCell), and 50 µg/ml Gentamycin. Before media collection, all cells were cultured in T75 flasks until they reached 70–80% confluency. Cells were washed with cDMEM without FCS and then cultured for 24 h in cDMEM without FCS until media collection. The cell culture supernatant was collected and pre-cleared from cells and larger particles by centrifugation at 400 × g at 4 °C for 10 min (Eppendorf 5810R) and 10,000 × g at 4 °C for 30 min (Eppendorf 5910R). Pre-cleared supernatants were subjected to ultracentrifugation at 29,000 rpm using a Beckman SW40 Ti rotor (RCF_avg: 106,154; RCF_max: 149,576; k-factor: 260) at 4 °C for 2 h. The dUC pellets were stored at -20 °C until further processing. The cells were trypsinized, washed in PBS, and cell lysates were prepared using RIPA buffer and stored at -20 °C until further processing. Experiments were performed once for LN18 and LN229, twice for HA and three times for NCH82.

Normal human astrocytes (ZY-1028) were purchased from ScienCell Research Laboratories (CA, USA) and cultured in astrocyte medium (ScienCell, CA, USA) supplemented with 10% fetal bovine serum (ScienCell, CA, USA), Astrocyte Growth Supplement (ScienCell, CA, USA) and 1% penicillin-streptomycin solution (ScienCell, CA, USA). Human GBM cell lines (A172, T98G, U251, U373, and LN229) and human embryonic kidney 293T (HEK-293T) cells were purchased from the Shanghai Institutes for Biological Sciences Cell Resource Center and cultured in Dulbecco’s modified Eagle’s medium (HyClone, UT, USA) supplemented with 10% fetal bovine serum (Gibco, NY, USA). All cells were authenticated by Short Tandem Repeat (STR) with Cell Line Authentications and maintained in an incubator at 37 °C and 5% CO₂.

Primary human astrocytes (ScienCell Research Laboratories, Carlsbad, CA) were grown to confluence in Basal Medium Eagle (Thermo Fisher Scientific, Waltham, MA) buffered to pH ranging from 7.2–7.5 with 2.2 g/L sodium bicarbonate and 15 mM HEPES (Gibco, Grand Island, New York). Media was supplemented with 2% fetal bovine serum (FBS) (R&D Systems, Minneapolis, MN), 1% penicillin–streptomycin 10,000U/mL (Gibco), and 1% astrocyte growth supplement (ScienCell Research Laboratories). Astrocytes were used at passages 3–4 for all experiments.
Primary human brain microvascular endothelial cells (Cell Systems, Kirkland, WA) were grown to confluence on tissue culture plates coated with 0.2% gelatin (Thermo Fisher Scientific) in medium 199 (M199) (Gibco) buffered to pH ranging from 7.2–7.5 with 2.2 g/L sodium bicarbonate and 15 mM HEPES (Gibco). Complete M199 media (M199C) was comprised of 20% heat-inactivated newborn calf serum (Gibco), 1% penicillin–streptomycin 10,000U/mL (Gibco), 25 mg/L heparin (Sigma, St. Louis, MO), 5% heat-inactivated human serum AB (GeminiBio, Sacramento, CA), 50 mg/L ascorbic acid (Sigma), 7.5 mg/L endothelial cell growth supplement (Sigma), 2 mM L-glutamine (Gibco), and 5 mg/L bovine brain extract (Lonza, San Diego, CA). Endothelial cells were used at passages 9–16 for all experiments.

Astrocytes isolated from human brainstem (HA-bs; Sciencell) were cultured in basal astrocyte medium supplemented with 2% FCS, 1% astrocyte growth supplement and 1% penicillin–streptomycin (all components from Sciencell).

Primary human astrocytes were purchased from ScienCell Research Laboratories (Carlsbad, CA; Cat. # 1800-5). These cells were grown on the astrocyte medium purchased from ScienCell laboratories (Cat. # 1801) containing 2% of fetal bovine serum (ScienCell Cat. # 0010), astrocyte growth supplement (ScienCell Cat. # 1852) and penicillin/streptomycin (ScienCell Cat. # 0503), antibiotic/antimycotic solution (Sigma-Aldrich, St. Louis, MO, United States) (Atluri et al., 2013 (link)).