Imager software version 3

Cell count was determined by quantifying nuclei per images, following DAPI staining. This task was performed automatically using Lionheart FX automated microscope using GEN5 Microplate Reader and Imager Software Version 3.05 for imaging and quantification, both from BioTek Instruments Inc. (Vermont, USA). Additionally, we determined cell viability via biomass performing a crystal violet assay using a protocol previously described (Del Favero et al. 2021b (link)). Briefly, cells were rinsed with warm DPBS before fixation with cold ethanol (99%). Staining was performed for 5 min using crystal violet solution (0.1%). Cells were washed 4 times using autoclaved water. Cells were lysed for 10 min on an orbital shaker (500U/min) using 99% EtOH and acetic acid (1%), before measuring absorbance [595 nm; Cytation 3 multi-mode plate reader (BioTek Instruments, Winooski, VT, United States)]. Three independent cell preparations were prepared and three technical replicates were measured.

For migration assay, 140,000 T24 cells were seeded with 2 ml cell culture medium in 35 mm 6-well plates (Sarstedt REF 83.3920.005). The assays were performed 48 h after the seeding, at this point almost full confluence was achieved. Scratching was done using a 200 µL pipette tip, followed by a washing step with medium. The tip was changed every 3 wells. After addition of 2 ml treatment solution, pictures were taken on Lionheart FX automated microscope using GEN5 Microplate Reader and Imager Software Version 3.05 for imaging and quantification both from BioTek Instruments Inc. (Vermont, USA). After 24 h incubation at 37 °C and 5% CO_2, a secondary set of pictures was taken at the same coordinates. Quantification of the healed area was performed using ImageJ 1.53a software. 3 experimental replicates were performed and at least 3 optical fields were evaluated, resulting in at least 9 optical fields per condition.