Gen5 microplate data collection and analysis software

To further comprehend the effects of nicotine induced in the selected cells in the present study, the morphology of the cells was evaluated after 24 h of stimulation. A microscopic evaluation was carried out by photographing the cells under bright-field illumination. The pictures were analyzed using Gen5™ Microplate Data Collection and Analysis Software (BioTek Instruments Inc., Winooski, VT, USA).

The wound healing (scratch) test was used to assess the effect of Met on cell migration. For this purpose, the cells were cultured in 24-well Corning plates (1 × 10⁵ cells/ well) and an automatic scratch was made in the middle of each well using the AutoScratch ™ Wound Making Tool provided by BioTek^® Instruments Inc., Winooski, VT, USA as recommended by the manufacturer. Cells were then stimulated with the five Met concentrations (0.5, 1, 1.5, 2 and 2.5 mM) for a 24-hour interval. Photographs (4 × magnification) were taken both at the beginning of the experiment (0 h) and after the stimulation was completed (24 h) using Cytation 1 and were processed using Gen5 ™ Microplate Data Collection and Analysis Software (BioTek^® Instruments Inc., Winooski, VT, USA). The effect of Met on cell migration was quantified by calculating the migration rate, expressed as a percentage, using the formula described above [19 (link)].

For the immunofluorescence visualization of the cellular components, HepaRG was cultured in plates of 12 wells, with 1 × 10⁵ cells / well, and after reaching a corresponding confluence of approximately 90%, they were stimulated for 72 h with Met (0.5, 1, 1.5, 2 and 2.5 mM). After 24 h, the cells were washed with ice-cold PBS and then fixed with paraformaldehyde 4% by maintaining room temperature for one hour. Cell permeabilization was performed with Triton X 2% in PBS. Then, 30% FBS solution in 0.01% Triton X was used for one hour to block Triton X 2% action. The cells were incubated at 4 °C overnight with Anti-COX IV antibody Mitochondrial marker (ab33985) at a dilution. of 1:500 for mitochondrial visualization. The next day, the primary antibody was washed with 0.01% Triton X solution in PBS and the secondary antibody specific for COX IV mitochondrial marker—Donkey Anti-Goat IgG H&L (Alexa Fluor^® 488—ab150129) was added, and the plate was kept for 2 h at room temperature and protected from light. After this time, 4′, 6-diamidino-2-phenylindole (DAPI) staining was added to visualize the nuclei. The pictures were then taken with Cytation 1 and processed using Gen5 ™ Micro-plate Data Collection and Analysis Software (BioTek^® Instruments Inc., Winooski, VT, USA).

To verify the impact of PCs (RUT, OA and LA) and REs (RUT-O and RUT-L) on the morphology and confluence of H9c2(2-1), HaCaT, and HepaRG cells, a microscopic examination was performed. The cells were observed under bright field illumination and photographed at the end of the 24 h treatment period using Cytation 1 (BioTek Instruments Inc., Winooski, VT, United States). The photographs were processed using the Gen5™ Microplate Data Collection and Analysis Software (BioTek Instruments Inc., Winooski, VT, United States).

The potential toxicity of 100 μM RUT, OA, LA, RUT-O, and RUT-L at nuclear level was tested by applying the Hoechst 33342 staining assay protocol according to the manufacturer’s (Thermo Fisher Scientific, Inc., Waltham, MA, United States) recommendations. In brief, H9c2(2-1), HepaRG, and HaCaT cells were seeded in 12-well plates (10⁵ cells/1.5 ml/well) and treated with the test compounds for 24 h. After the stimulation period, the media was removed, and the staining solution diluted 1:2000 in PBS was added (500 µl/well). The plates were incubated for 10 min at room temperature, protected from light. Finally, the staining solution was washed with PBS and the pictures were taken using Cytation 1 (BioTek Instruments Inc., Winooski, VT, United States) and analyzed by the means of Gen5™ Microplate Data Collection and Analysis Software (BioTek Instruments Inc., Winooski, VT, United States). Staurosporine (STP) 5 µM was selected as positive control for apoptosis.

To determine the cytotoxic potential of the test samples, a microscopic evaluation of the cells’ morphology and shape was performed. The cells (10,000 cells/200 µL/well) were observed under bright field illumination and photographed at 24 h after treatment and compared with the solvent (media). The photos were taken using Cytation 1 (BioTek Instruments Inc., Winooski, VT, USA). The analysis of the images was performed by means of the Gen5™ microplate data collection and analysis software (BioTek Instruments Inc., Winooski, VT, USA).