B 27 supplement 50x

For a mammosphere forming assay by radiation-induced ROS, cells were seeded in 10 cm plates and were incubated for 24 h for attachment. Cells were incubated with serum-reduced (0.2% FBS) medium for 16 h for starvation and irradiated with 10 Gy with or without 30-min pretreatment with 100 nM vactosertib. After 24 h of incubation, we harvested the adherent cells with trypsin–EDTA and reseeded the cells in ultra-low attachment dishes. Cells were incubated in DMEM/F-12 (1:1) (GenDEPOT) supplemented with B-27 Supplement (50X) (Gibco Laboratories), 10 ng/ml basic fibroblast growth factor (bFGF) (Invitrogen), and 10 ng/ml Epidermal Growth Factor (EGF) (Sigma Aldrich). To measure mammosphere-forming efficiency (MSFE), the number of spheres (> 50 μm) was counted after one week. MSFE indicates the number of spheres divided by the original number of cells seeded and is presented as %.
For a mammosphere forming assay by GOX-generated ROS, cells were seeded in ultra-low attachment dishes and incubated in DMEM/F-12 (1:1) (GenDEPOT) supplemented with B-27 Supplement (50X) (Gibco Laboratories), 10 ng/ml bFGF (Invitrogen), and 10 ng/ml EGF (Sigma Aldrich). Cells were treated with 2.5 mU/ml GOX with or without co-treatment with 100 nM vactosertib. To measure MSFE, the number of spheres (> 100 μm) was counted after one week.

From naïve mice, all DRG were dissected aseptically and placed in Hibernate A (Gibco; Thermo-Fisher) supplemented with 0.5 mM L-Glutamine (Gibco; Thermo-Fisher) and 2% B-27 Supplement (50X) (Gibco; Thermo-Fisher). Dorsal root ganglia were dissociated in 2-mg/mL papain in Hank's balanced salt solution (HBSS) (30 minutes; 37°C & 5% CO₂) followed by 2.5-mg/mL collagenase in HBSS (30 minutes; 37°C & 5% CO₂). Cells were triturated and then resuspended in Neurobasal A (Gibco; Thermo-Fisher) supplemented with 0.5 mM L-Glutamine (Gibco; Thermo-Fisher) and 2% B-27 Supplement (50X) (Gibco; Thermo-Fisher). Cell suspensions were then seeded onto laminin-coated glass coverslips and incubated for 30 minutes (37°C & 5% CO₂) before overnight incubation in Neurobasal medium. For experiments involving pertussis toxin (PTX) before incubation, before calcium imaging, cells were incubated with 500-ng/mL PTX (EMD Millipore, Burlington, MA) in Neurobasal medium for 18 hours at 37°C, with controls incubated in Neurobasal medium plus sterile H₂O vehicle.

Three different culture media were tested to find the one that could best preserve tissue quality and secure cell survival. First medium: 65% DMEM (Gibco), 25% Hank’s solution (Gibco), 6.5 mg/mL glucose (Merck, Germany), 10% Antibiotic-Antimycotic solution (Gibco) (penicillin 10,000 units/mL, streptomycin 10,000 µg/mL and Amphotericin B 25 µg/mL). In the second medium, Hank’s solution and glucose were replaced for 25% B-27™ Supplement (50X) (Gibco) and 6.5 mg/mL D-glucose (Sigma-Aldrich) respectively.
The third medium was composed of 60% DMEM/F12 (Gibco), 30% Horse serum (Gibco/Lifetech), 10% Antibiotic-Antimycotic (Gibco), 6.5 mg/mL D-glucose (Sigma-Aldrich), 50 µL/10 mL of L-Glutamine 200 mM (Sigma-Aldrich). In addition, effects of BDNF 250 ng/mL (Hellobio) were evaluated, and all experiments at DIV 14 included this factor. All media were filtered with disposable cellulose acetate syringe filter units, 3 mm Filter Diameter/0.20 μm Filter Pore Size (Micro Filtration Systems) and prepared under aseptic conditions in a laminar flow cabinet.