Aqueous crystal violet solution

For the biofilm assay, overnight cultures of PM1 and PM2 were diluted to obtain a concentration of 10⁷ CFUs/mL, and aliquots (200 μL) of the diluted bacterial suspension were placed into 96-well, flat-bottomed, sterile polystyrene microplates (Costar, Corning, Inc., Corning, NY, USA), with or without Lactobacillus spp. at the same concentration, and incubated overnight at 37 °C. The biofilm formed was quantified by a modification of the crystal violet assay [35 (link)]. After 24 h, the attached bacteria were washed twice with 200 μL of PBS (Microgem, Naples, Italy) and air-dried for 45 min. The wells were then stained with 200 μL of 1% aqueous crystal violet solution (Sigma-Aldrich, Merck, Milan, Italy) for 45 min. The plates were rinsed with 200 μL of sterile distilled water to remove excess dye and air-dried. The dye associated with the attached biofilm was dissolved in a solution of 200 μL of 100% ethanol, and the OD_570/655 absorbance was measured on a microplate reader (Biorad, Hercules, CA, USA).

Compound effect on cell colony formation and growth was established by clonogenic assay, as previously published [45 (link)]. Briefly, MIA PaCa-2 and PANC-1 cells were seeded into 12-well plates (Corning) at a density of 200 cells/well in 1 mL of medium and treated with sunitinib derivatives at the concentrations representing 10% and 90% of calculated EC₅₀ value. Cells were incubated in a humidified atmosphere containing 5% CO₂ at 37 °C and grown for 8 days. After incubation, the cells were rinsed with PBS and fixed with 10% formaldehyde, methanol-free (ThermoFisher Scientific, Waltham, MA, USA) solution for 20 min. Then the cells were rinsed with PBS two more times, and cell colonies were stained with 0.1% aqueous crystal violet solution (Sigma-Aldrich Co.) for 20 min, followed by rinses with sterile deionized water to remove the excess dye. Photos of colonies were taken using the G:BOX gel documentation system (Syngene International Ltd., Bengaluru, India) and Genesys software (Syngene International Ltd.). The number of colonies and their area were calculated using GeneTools colony and cell counting software (Syngene International Ltd.) according to the manufacturer’s instructions. Cell colony area and number in control groups were normalized to 100%. The percentage of drug-treated colony area and number were compared to the control.