To establish and propagate aRMS cell spheres, we modified a protocol developed for eRMS cells (42 (link)). Briefly, aRMS cells were grown in ultra-low attachment plates or flasks (Corning) in Neurobasal media (Gibco) supplemented with 1X B27 [2X B27 for Rh28 cells] (Invitrogen), 80ng/ml bFGF (Corning), 40ng/ml EGF (Sigma), and 50μg/ml insulin. Limiting dilution assays were based on sphere formation, and assessed 48 wells per condition. Wells were scored positive (≥ 1 sphere/well) or negative (0 spheres/well) for sphere formation after seven days in culture. Sphere forming frequency and statistics were calculated using ELDA software (43 (link)).
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Propagation of aRMS Cell Spheres
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Corresponding Organization : Duke University
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Variable analysis
independent variables
- Cell type (aRMS cells)
- Culture conditions (ultra-low attachment plates or flasks, Neurobasal media, B27 supplement, bFGF, EGF, insulin)
- Sphere formation (scored as positive or negative after seven days in culture)
- Sphere forming frequency
- Culture duration (seven days)
- Limiting dilution assay (48 wells per condition)
- Positive control: eRMS cells (as described in the cited protocol)
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