Vi cell 12 sample carousel cell viability analyzer

Immortalized human Schwann cell (HSC1λ) and immortalized neurofibroma cell (ipNF06.2A^{30 (link)}) were obtained from Dr. Margaret Wallace’s laboratory at the University of Florida. MPNST cell lines (STS-26T^{63 (link)}, S462^{64 (link)}, S462TY, T265^{65 (link)}) were obtained from Dr. David Largaespada’s laboratory at the University of Minnesota. All cell lines were cultured with Dulbecco’s modified Eagle’s medium (DMEM, Wisent Technologies) supplemented with 10% fetal bovine serum (FBS, Wisent Technologies) and 1% penicillin/streptomycin. Cells were grown at 37 °C and 5% CO₂. For direct cell counting, 1 × 10⁵ cells were plated in triplicates into 6 well plates in 2 ml of medium. After incubation times (days 1–5), cells were collected and analyzed for cell count and cell viability. Cells were directly counted using Trypan blue and the Beckman Coulter Vi-CELL (12-sample carousel) Cell Viability Analyzer (Beckman Coulter). IC50 assays were performed in 96 wells by seeding 5000 cells in triplicate overnight. Cells were treated with sonidegib (NVP-LDE225, Selleck Chemicals) the following day with increasing drug concentrations and read by CellTitre-Glo luminescent cell viability assay in accordance with the manufacturer’s instructions (Promega, G7570) on a 96-well plate reader (GloMax-96 microplate luminometer; Promega).

Equal numbers of cells were plated in triplicate in 96-well plates and cellular proliferation was assessed using Alamar Blue proliferation assay as per manufacturer’s instructions (Invitrogen) or 5-bromodeoxyuridine cell proliferation assay as per manufacturer’s instructions (BioVision). Trypan blue exclusion assay was performed by directly counting cells plated in triplicate using the Beckman Coulter Vi-CELL (12-Sample Carousel) Cell Viability Analyzer (Beckman Coulter). For drug sensitivity assays 5000 cells were seeded in 96-well plates and 24 h later the cells were treated with increasing concentrations of drugs and Alamar Blue assay was performed. Additionally, 24 h post transfection with indicated plasmids cells were labeled with Cell Proliferation Dye eFluor 670 (eBioscience) per manufacturer’s instruction. At indicated time points, dilution of the dye, and hence, proliferation was assessed by flow cytometry at the Core Flow Cytometry Facility of the Hospital for Sick Children. All the proliferation assays were repeated at least in triplicates. To assess neurosphere formation ability, GSCs were seeded at a density of 1000 cells per well in 6-well plates in triplicate and the resulting spheres were counted 10–14 days later.