2.5.1. Tetrazolium Salt (MTS) Cell Viability Assay in 2D Culture

NB cell lines and healthy control cells (HA-CAT and B-end) were seeded in 96-well plates (4−9.6 × 10³ cells/well, depending on the cell line used). The day after seeding, cells were treated with OLE, in quadruplicate for each experimental condition, as already detailed above. After 48–120 h, cells were processed to determine the cytotoxic effects mediated by OLE treatment. The MTS tetrazolium compound assay was used, according to the manufacturer’s instructions (CellTiter 96^® Aqueous One Solution Cell Proliferation Assay, Promega Italia, Milano, Italy). The MTS tetrazolium compound is converted by metabolically active cells to produce a colored formazan, whose amount is proportional to the number of viable cells. In some experiments, NB cell lines and healthy control cells were treated with OLE for 18 h (namely “short-treatment” experiments). Then, OLE was removed, and cells were recovered with fresh complete medium for an additional 54 and 102 h. At the end point time, 72 and 120 h respectively, the MTS assay was used.

In a separate set of experiments, NB cells seeded as described above were treated with a combination of OLE and topotecan (TOPO, Sigma-Aldrich, St. Louis, MO, USA). The doses of TOPO were chosen on the basis of the literature [25]. After 96 h of treatment, cells were subjected to the MTS assay to determine cytotoxic effects. Then, the results were analyzed by the use of the Compusyn online software (available at http://www.combosyn.com (accessed on 10 June 2021)/, Combosyn, Inc., Paramus, NJ, USA), with the aim to assess synergism, addiction, and/or antagonism between OLE and TOPO.