Evaluation of spheroid viability

The reduction of Resazurin by metabolically active cells was the method used to assess viability (measured with 10% solution of Uptiblue®). Fluorescent intensity was then measured after 24 hours with spectrofluorimetry (Fusion®, Packard Bioscience, USA) using 530–560 nm excitation wavelength and 590 nm emission wavelength. All experiments were done in triplicate. Flow cytometric analysis for the viability assay was performed independently for both types of cells on a Fortessa® flow cytometer (BD®, Becton, Dickinson and Company, USA). The US CI 20 was selected for cytometer experiments of US-treated spheroids: after treatment, six spheroids were collected in an assay tube and dissociated using 1 ml trypsin solution (0,25% Trysin-EDTA, Gibco®, Thermofisher scientific). Cells were then marked with the BV-510 viability marker. FSC and SSC, providing green and red fluorescence signals at 530 and 650 nm, respectively, and BV-510 marker were evaluated for each cell. A total of 10,000 events were recorded for each tube. All experiments were performed four times with six dissociated KPCF spheroids per condition. Measurements were done under fixed instrument settings. Results were analyzed using Flowjo® software (LLC, USA).