4.5.1. Cells Treatment before and after Exposure to the Extract

The TE671 rhabdomyosarcoma cancer cell line was obtained from the European Collection of cell cultures (ECACC, London, UK). The A172 glioblastoma cell line was obtained from a male patient of 53 years old (ECACC, London, UK, Cat. Nr 88062428). Cells were grown in a cell-culture flask (75 cm<sup>2</sup> surface area) in Dulbecco's modified Eagle's Medium (DMEM) (ThermoFisher Scientific Inc. (Gibco), Waltham, MA, USA Cat. Nr. 10566016) enriched with glucose (4500 mg/mL), and 15% fetal bovine serum (FBS) (ThermoFisher Scientific Inc. (Gibco), Waltham, MA, USA Cat. Nr. 26140-079), L-glutamine (2 mM) (ThermoFisher Scientific Inc. (Gibco), Waltham, MA, USA Cat. Nr. A12860-01). A dual antibiotic solution of penicillin G (100IU) and streptomycin (100 μg/mL) was added (ThermoFisher Scientific Inc. (Gibco), Waltham, MA, USA Cat. Nr. 15140-122), in addition to an amphotericin B solution (ThermoFisher Scientific Inc. (Gibco), Waltham, MA, USA Cat. Nr. 14140-122). A Coulter counter apparatus was used to measure the number of cells inoculated in the experimental setup. Cells were seeded in 96-well plates (1.5 × 103 cells/mL) and were allowed to grow for 24 h until reaching ~80% confluence. After 24 h, cells were exposed to successive diluted concentrations of the extract (t = 0 h), ranging from 0.04 to 6.25 mg/mL, derived from the dried extract diluted de novo in DMSO (10% *v*/*v*). The final concentration of DMSO when the extract was added to cell culture was 1% *v*/*v*. A control well with the same concentration of DMSO was used to confirm that no toxic effect was observed. Cells were incubated for 24, 48 and 72 h.

Experiments were performed in 96-well plates (CellStar® Sigma-Aldrich Chemie GmbH, Taufkirchen, DE Cat. Nr. M3687-60EA, Saint Louis, MO, USA). Plate set up was as follows: a column contained only cell culture medium, a column of cell culture and the staining chemical, a column with cultured cells and a column with cultured cells plus the staining chemical. The remaining wells were used for the testing of the extract in various concentrations. As blank were used those wells containing cell culture medium only, cells and no staining agent or drug, whereas as positive control were used those wells with cultured cells without staining agent. All experiments were performed in triplicate.
