Glioblastoma Cell Culture Protocol

PIGPCs were isolated as previously described [39 (link)] and grown in DMEM (Life Technologies) with 10% fetal bovine serum (FBS) and 1% PenStrep solution (Corning). U3046MG, U3035MG, U3082MG, U3084MG, and U3065MG cells were obtained from the Human Glioblastoma Cell Culture Resource (HGCC) and cultured as described previously [23 (link)] in Neurobasal (GIBCO) and DMEM/F12 with Glutamax media (Life Technologies), 1:1 mix, with 1% PenStrep, N2 and B27 (Life Technologies), 10 ng/mL epidermal growth factor (EGF), and 10 ng/mL fibroblast growth factor (FGF) (Peprotech). Cells were dissociated by Accutase (ThermoFisher) treatment, and grown as a monolayer on plastic dishes coated with polyornithine (Sigma) and laminin (Biolamina). U251MG and T98G cells were obtained from ATCC and grown in DMEM with 10% FBS and 1% PenStrep solution. Cells were used within ten passages. Mycoplasma contamination was tested every 3 months.
Hypoxia (1%O₂) was generated in a Whitley H35 Hypoxystation (Don Whitley Scientific, Bingley, UK).
Reagents: TAPI-2 (Sigma), MI1 (Tocris Bioscience), LY294002 (Sigma), added 24 h pre-hypoxia exposure.

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Grassi E.S., Pantazopoulou V, & Pietras A. (2020). Hypoxia-induced release, nuclear translocation, and signaling activity of a DLK1 intracellular fragment in glioma. Oncogene, 39(20), 4028-4044.

Publication 2020

DMEM PenStrep solution Neurobasal Accutase polyornithine laminin Whitley H35 Hypoxystation TAPI-2 LY294002

Accutase Cell culture Cells Dishes Epidermal growth factor Fetal bovine serum Glioblastoma Human resource Hypoxia Laminin Ly294002 Mycoplasma Polyornithine Tapi 2

Corresponding Organization :

Other organizations : Lund University

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Variable analysis

independent variables

Hypoxia (1%O2) generated in a Whitley H35 Hypoxystation
TAPI-2 (Sigma)
MI1 (Tocris Bioscience)
LY294002 (Sigma)

dependent variables

Not explicitly mentioned

control variables

PIGPCs isolated and grown in DMEM with 10% FBS and 1% PenStrep solution
U3046MG, U3035MG, U3082MG, U3084MG, and U3065MG cells obtained from the Human Glioblastoma Cell Culture Resource (HGCC) and cultured in Neurobasal and DMEM/F12 with Glutamax media, 1:1 mix, with 1% PenStrep, N2 and B27, 10 ng/mL EGF, and 10 ng/mL FGF
U251MG and T98G cells obtained from ATCC and grown in DMEM with 10% FBS and 1% PenStrep solution
Cells used within ten passages
Mycoplasma contamination tested every 3 months

controls

Positive controls not specified
Negative controls not specified

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