Normal brainstem was isolated from Ntv-a or Ntva;p53fl/fl postnatal day 3 (P3) pups and was digested and dissociated as described above for Olig2-eGFP-L10a tumors. Cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin at 37°C and 5% CO2, and passaged a maximum of 3 times for experiments. Cells were plated in clear 6-well plates for Annexin V assays, clear 96-well plates for BrdU assays, and white-walled, clear-bottomed 96-well plates for Caspase 3/7 assays.
To concentrate RCAS viruses, DF1 cells transfected with RCAS plasmids were passaged a minimum of 6 times from transfection, and then passaged 1:12. After 3 days, virus-containing media was harvested, centrifuged to remove cell debris, filtered through 0.45 μm pores, and concentrated 100-fold using Retro-X Concentrator (Clontech) per the manufacturer’s instructions.
Brainstem progenitors were plated and infected with RCAS viruses at 1:100. Assays were conducted 3–5 days post-infection, or were split when confluent and subsequently plated for assays. For BrdU assays, the Cell Proliferation ELISA, BrdU Colorimetric kit (Roche) was utilized, per the manufacturer’s instructions using a Molecular Devices Versa Max Tunable Microplate reader. Caspase 3/7 activity was measured using the ApoToxGlo Triplex assay (Promega) per the manufacturer’s instructions using a Turner Biosystems Modulus Microplate Reader. To measure the percentage of cells staining positive for Annexin V, the FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen) was utilized per the manufacturer’s instructions. For cell counting experiments, 50,000 infected cells were plated in 6 well plates in duplicate. For each time point, 2 wells of each line were trypsinized and counted with a Sceptor 2.0 Cell Counter (Millipore). All experiments were done a minimum of three times on at least three independent preparations of progenitor cells.
To concentrate RCAS viruses, DF1 cells transfected with RCAS plasmids were passaged a minimum of 6 times from transfection, and then passaged 1:12. After 3 days, virus-containing media was harvested, centrifuged to remove cell debris, filtered through 0.45 μm pores, and concentrated 100-fold using Retro-X Concentrator (Clontech) per the manufacturer’s instructions.
Brainstem progenitors were plated and infected with RCAS viruses at 1:100. Assays were conducted 3–5 days post-infection, or were split when confluent and subsequently plated for assays. For BrdU assays, the Cell Proliferation ELISA, BrdU Colorimetric kit (Roche) was utilized, per the manufacturer’s instructions using a Molecular Devices Versa Max Tunable Microplate reader. Caspase 3/7 activity was measured using the ApoToxGlo Triplex assay (Promega) per the manufacturer’s instructions using a Turner Biosystems Modulus Microplate Reader. To measure the percentage of cells staining positive for Annexin V, the FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen) was utilized per the manufacturer’s instructions. For cell counting experiments, 50,000 infected cells were plated in 6 well plates in duplicate. For each time point, 2 wells of each line were trypsinized and counted with a Sceptor 2.0 Cell Counter (Millipore). All experiments were done a minimum of three times on at least three independent preparations of progenitor cells.
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