Ultra low attachment plates or flasks

RD and Rh36 rhabdospheres were cultured as described (52 (link)). A protocol to culture SMS-CTR spheres was developed, in which sphere media was supplemented with 4× bFGF, 4× EGF, and 50µg/ml insulin. This protocol was also used for the JR-1, CCA, and CT-TC cell lines since they had not previously been cultured as rhabdospheres. Sphere experiments were performed in either 96-well, 6-well, 10cm, or 25ml ultra-low attachment plates or flasks (Corning). All conditions were plated at the same cell density and spheres were allowed to form over 48hrs before genetic or pharmacologic manipulation. To induce shRNA or cDNA expression, spheres were manually dissociated and treated with 4µg/ml (RD) or 5µg/ml (SMS-CTR) doxycycline (dox) daily for 4 days. Unless noted, all shRNA experiments utilized the dox-inducible system. At the end of experiments, spheres were photographed and collected. Spheres were measured and quantified using Image J (NIH), 4 photographs per condition. The length and width of each sphere was measured and averaged, then spheres were categorized into four groups (<0.2mm, 0.2–0.5mm, 0.5–1mm, and >1mm in diameter) based on a neurosphere protocol (53 ).

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)).