Pmg8041

The in vitro neurosphere assay was performed to assess the numbers of neural stem cells, as previously described [8 (link)]. Briefly, the periventricular region was dissected; cells were mechanically dissociated and placed in an enzyme solution containing 1.33 mg/ml trypsin (T1005, Sigma-Aldrich), 0.76 mg/ml hyaluronidase (H6254, Sigma-Aldrich) and 0.13 mg/ml kynurenic acid (K3375, Sigma-Aldrich) in artificial cerebrospinal spinal fluid [8 (link)]. Cells were incubated for 25 min at 37 °C, followed by centrifugation for 5 min at 1500 rpm. The supernatant was removed, and samples were placed in a trypsin inhibitor solution consisting of 0.67 mg/ml ovomucoid (LS003086, Worthington, Lakewood, NJ, USA) in serum-free media (SFM) containing l-glutamine (2 mM; Invitrogen, Waltham, MA, USA) and penicillin/streptavidin (100 U/0.1 mg/ml; Invitrogen) [8 (link)]. Samples were triturated and centrifuged for 5 min at 1500 rpm, then added to SFM supplemented with epidermal growth factor (20 ng/ml; PMG8041, GIBCO, Pittsburgh, PA, USA), basic fibroblast growth factor (10 ng/ml; PHG0266, GIBCO) and heparin (2ug/ml; H3149, Sigma-Aldrich), plated at 10 cells/ul (Tabake-Coles et al. 2008) and incubated for 7 days at 37 °C and 5% CO₂. The numbers of neurospheres (> 80um in diameter) were counted.

NSC neurospheres were obtained from control (injured) C57BL/6 periventricular dissections, as described above. Neurospheres were collected and mechanically disassociated into single cells and plated in 96-well plates coated with 4% Matrigel (Corning 354234, Corning, NY, USA) at a density of 20,000 cells per well in 100 µL of SFM with 20 ng/µL epidermal growth factor (GIBCO, PMG8041, Waltham, MA, USA), 10 ng/µL basic fibroblast growth factor (GIBCO, PHG0266, Waltham, MA, USA) and 2 µg/mL heparin (Sigma-Aldrich, H3149, St. Louis, MO, USA) (referred to as growth factors). One day later, the media was removed and replaced with fresh SFM, growth factors, 20 µM cisplatin (Sigma-Aldrich, P4394, St. Louis, MO, USA) and 10 µM NWL283 (New World Laboratories, Laval, QC, Canada) in sterile PBS. The vehicle group received media containing SFM, growth factors and PBS.
To assess cell viability at 2 h and 8 h, cells were fixed with 4% PFA for 15 min and washed three times for 5 min in PBS. Cells were incubated for 10 min with 1:10,000 DAPI (Invitrogen, D130, Waltham, MA, USA), then washed three times for 5 min in PBS and imaged on an Axio Observer Zeiss microscope. DAPI cells were counted using ImageJ (https://imagej.net/ij/) analyze particle detection and then manually confirmed. Data were collected from three independent experiments.