Sphere-forming cells were obtained as previously described (Ciccarelli et al., 2016 (link); Camero et al., 2020 (link); Megiorni et al., 2021 (link)). Briefly, RD and RH30 cells were cultured in anchorage-independent conditions (ultra-low attachment flasks or plates, Corning) in stem cell (SC)-medium consisting of DMEM:F12 medium (Gibco-Invitrogen) and B27 (ThermoFischer). Fresh human epidermal growth factor (20 ng/ml) and fibroblast growth factor (20 ng/ml) (PeproTech, London, United Kingdom) were added twice/week until cells formed floating spheres. To evaluate the primary sphere formation, cells from sub-confluent (70–80%) monolayer cultures were plated at a density of 100, 500 or 1,000 cells in a 24-well culture plate (Corning Inc., Corning, NY, United States). For the sphere formation assay, the number of primary tumor spheres was determined.
Vaccaro S., Rossetti A., Porrazzo A., Camero S., Cassandri M., Pomella S., Tomaciello M., Macioce G., Pedini F., Barillari G., Marchese C., Rota R., Cenci G., Tombolini M., Newman R.A., Yang P., Codenotti S., Fanzani A., Megiorni F., Festuccia C., Minniti G., Gravina G.L., Vulcano F., Milazzo L, & Marampon F. (2022). The botanical drug PBI-05204, a supercritical CO2 extract of Nerium oleander, sensitizes alveolar and embryonal rhabdomyosarcoma to radiotherapy in vitro and in vivo. Frontiers in Pharmacology, 13, 1071176.
Other organizations :
Policlinico Umberto I, Sapienza University of Rome, Istituto Superiore di Sanità, University of L'Aquila, Bambino Gesù Children's Hospital, University of Rome Tor Vergata, Istituto Pasteur, Azienda Ospedaliera San Giovanni Addolorata, The University of Texas MD Anderson Cancer Center, University of Brescia, Istituto Neurologico Mediterraneo, University of Siena, San Salvatore Hospital
No positive or negative controls were explicitly mentioned.
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