Penstrep solution

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.

After the mice were euthanized, the spinal column was opened. Ten to twelve DRG were excised from the vertebral level where TDI was applied and enzymatically digested in 2.5 mg/ml dispase II (Sigma-Aldrich, Taufkirchen, Germany) and 2.5 mg/ml collagenase type I (Alfa Aesar, MA, USA) in Ca²⁺ and Mg²⁺⁻free Hank’s Buffered Salt Solution (Coutiva, Utah, USA) for 60 min at 37 ºC. The cell solution was dissociated by trituration using Pasteur pipettes. Cells were washed in DRG medium (Dulbecco’s modified Eagle medium, Corning, VA, USA) with 10% fetal bovine serum (FBS, Biowest, Nuaillé, France) and Pen-strep solution (100 U/mL streptomycin and 100 U/mL penicillin (Corning, VA, USA)) by centrifugation for three times. The cell pellet was resuspended in 120 µl DRG medium and placed onto 0.1 mg/ml poly-L-lysine (Sigma-Aldrich, Taufkirchen, Germany) and 5 µg/ml laminin (Roche, Mannheim, Germany)-coated coverslips (20 µl cell suspension per a coverslip). The DRG neurons were incubated at 37 °C for 2 h and then flooded with an additional 1 ml DRG medium and further incubated at 37 °C for 16 h before performing calcium imaging or co-culture study [11 (link)].