96 well flat bottom cell culture plate

For this assay, wells of a 96-well flat-bottom cell culture plate (Greiner Bio-one) were filled with 180 μl of pH and sodium adjusted LBB⁻ media. Again, the tested pH values were 6.5, 7.5, and 8.5, and sodium concentrations ranged from zero to 500 mM NaCl added. Cultures were re-suspended to an OD₆₀₀ of 0.1 and 20 μl was added to each respective well in duplicates for each condition. Using an iMark plate reader (BioRad), the optical density at OD₅₉₅ was recorded for each well after a 24-h incubation at 37 °C (no shaking). Then, the media was discarded and the plates were washed three times with deionized water and left to dry at room temperature. One hundred microliters of an 0.1% (w/v) crystal violet stain was added to each well and shaken at 7×g and 37 °C in an orbital shaker (Thermo Scientific MaxQ 4000) for 30 min. After staining, the plates were washed three times with deionized water and left to dry at room temperature. Then, 200 μl of 95% ethanol was added to the wells to solubilize the stained biofilm and consequently incubated for 15 min at room temperature. Optical density at 570 nm was measured in the iMark plate reader to assess the biofilm formation, and the biofilm ratio (OD₅₇₀/OD₅₉₅) was calculated to account for growth deficiencies [1 (link)]. These experiments were independently repeated five times for each pH.

The CTC-MCC-41 line was treated as described above and seeded in technical triplicates in a density of 3000 cells per well into a 96 well flat bottom cell culture plate (Greiner Bio-One, Kremsmünster, Austria). The cells were allowed to settle down at 37 °C, 5% CO₂ overnight. The next day, the plate was placed into the IncuCyte^® Zoom Live Cell Analysis System (Essen Bioscience, Ann Arbor, MI, USA) and cell confluence was measured every 2 hours. Analysis of data was performed based on cell confluence using the provided manufacturer’s IncuCyte^® Zoom Software. Statistical analysis of acquired data was carried out using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). For the therapeutic drug sensitivity testing the CTC-MCC-41 line was prepared as written above for the proliferation assay. The next day, freshly prepared serial dilutions of either AKT inhibitor MK2206 (#S1078, Selleck Chemicals, Houston, TX, USA), mTOR inhibitor RAD001 (#S1120, Selleck Chemicals, Houston, TX, USA), the combination of both drugs or vehicle dimethyl sulfoxide (DMSO) (#D5879, Sigma-Aldrich, St. Louis, MO, USA) were added to the cells. Measurement and calculation using the IncuCyte^® Zoom system was carried out as described in the proliferation section above.

All wells of a 96-well flat-bottom cell culture plate (Greiner Bio-one) were filled with 140 μl of LBB⁻ media of pH 6.5 and increasing sodium concentrations of 0, 100, 200, 300, 400, and 500 mM in technical duplicates throughout the 12 well columns. Overnight cultures of each strain were pelleted by centrifugation at 3800×g for 5 min, washed with LBB⁻, and re-suspended to an optical density of 0.1 measured by wavelength 600 nm (OD₆₀₀). Twenty microliters of each culture was transferred to their respective wells, while control wells received 20 μl of sterile broth. All wells were then covered with 50 μl of mineral oil to prevent evaporation without affecting growth [7 (link)] and incubated at 37 °C and medium intensity shaking in a BioTek SynergyMx microplate reader for 24 h. The OD₅₉₅ was measured hourly for during that time. The plate setup and the measurements were repeated for pH 7.5 and 8.5 and a total of three biological replicates per pH.