24 well tissue culture test plates

The cell viability assay was based on the exclusion of propidium iodide (PI) by the intact viable cells according to the protocol described in [27 (link)]. The cells were plated in 24-well tissue culture test plates (Techno Plastic Products, Trasadingen, Switzerland) at 5 × 10⁴ cells/mL and treated with QBAs for 48 h. After incubation, PI (1 μg/mL) was added and the percentage of dead (PI-positive) cells was detected using Cytomics FC 500 and/or CytoFlex flow cytometry systems (Beckman Coulter, Inc., Brea, CA, USA). A total of 10,000 cells were analyzed for each sample. Each experiment included two replicate wells and was repeated at least three times.

Human osteosarcoma cells (SaOs2) were cultured in McCoy’s 5A medium (Gibco, Waltham, USA) supplemented with 15% fetal bovine serum (FBS) (Gibco, Waltham, MA, USA) and 1% penicillin (10,000 U/mL)-streptomycin (10000 µg/mL) (PEN-STREP) (Gibco, Waltham, MA, USA). The cells were split at passage P19 and seeded on the coupons placed in 24-well tissue-culture test plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland) at a density of 15000 cells/sample. They were cultured in a 5% CO₂ humid atmosphere at 37 °C for 1, 3, and 7 days. Glass coverslip (CS) of 12 mm diameter was used as an experimental control. The cells cultivated on CS were routinely visualized by using a Leica DMi1 (Leica Microsystems, Wetzlar, Germany) inverted phase contrast microscope.