HaCaT cells were obtained from CLS (Cell Line Services, Eppelheim, Germany) cell bank and were grown in Dulbecco's Modified Eagles Medium (DMEM) (Cytiva, Utah, USA) containing 10% Fetal Bovine Serum (Sigma Aldrich, Germany), 1% Penicillin streptomycin (Sigma Aldrich, Germany) and 7.5% NaHCO3 (AppliChem, Darmstadt) in a CO2 incubator at 37ºC until it reached 70% density.
Before being treated with BCAN, HaCaT cells were seeded into each well of a 96-well plate (TPP, Techno Plastic Products AG, Germany) at a density of 1x104 cells/well in 100 l of medium containing 10% FBS (LGC DR EHRENSTORFER, Germany). 100,000 M of methanol was used to dissolve 50 mg of the BCAN compound (CARLO ERBA, S.A.S.). Then, sterile distilled water was used to create 10,000 M BCAN master stock. Fresh preparations of 100 M of the material from the master stock were made in medium containing 10% FBS. The dosages used for the investigation were created via dilution from a 100 M drug. Six different concentrations of BCAN (5-10-20-40-60-80 M) were applied to HaCaT cells for 24 and 48 hours; eight duplicate wells per concentration were used, and the process was repeated in triplicate at various times. Controls for the untreated media (blank) and the solvent (methanol at a final concentration of 0.1%, v/v) were also tested concurrently. BCAN's cytotoxicity was assessed by measuring cell viability using the MTT and LDH tests. The MTT test was carried out as described by Mossmann in the past (1983). Microplate reader was used to detect absorbance at 570 nm (Synergy HTX, BioTEK, USA).
The LDH Cytotoxicity Detection Kit was used to perform the lactate-dehydrogenase (LDH) test (Roche, Germany). The supernatant was collected after treatment with BCAN concentrations, and the test was completed in accordance with the precise instructions included with the kit. To determine the released LDH enzyme, the OD values of the plates were measured at a 492 nm wavelength using a microplate reader (Synergy HTX, BioTEK, USA). There were three times and eight repeats for the experiments. Methanol was employed as a solvent control, with a final concentration of 0.1% (v/v).
The quantity of reactive oxygen species (ROS) in the cell was quantified using the Biovision kit (K936-250) (Milpitas, CA, USA). One x 104 cells were planted in each well of a 96-well black/clear-bottom plate (Greiner Bio-One GmbH, Germany). The media was taken out after 23 hours of waiting for the cells to adhere. Each well received 100 L of the ROS test buffer, and the cells were then washed in accordance with the kit's instructions. Following that, the wells were incubated for 40 minutes at 37°C with 100 L of 1X ROS marker made in ROS assay buffer. The 1X ROS marker was then removed, newly produced BCAN material was added to the wells in 5, 10, 20, 40, 60, and 80 M concentrations, and the plates were incubated for 24 hours at 37°C. After the incubation time, the ROS levels in the cell were determined fluorometrically in a microplate reader (Synergy HTX, BioTEK, USA) at 495/529 nm, and the outcomes were compared to the control group.
Utilizing the statistical package for social sciences' one-way analysis of variance (ANOVA) and Tukey's test, the results of the MTT, LDH, and ROS tests were assessed (SPSS). According to the percentages of control, the experimental research findings shown in the figures were reported as the mean standard deviation. By using the Tukey test, asterisks indicate a significant difference from the control group (p 0,005).
HaCaT cells (5x105) were planted on six-well cell culture plates (TPP, Techno Plastic Products AG, Germany) and allowed to develop until 70–80% confluence as a monolayer for the scratch wound healing experiment. Using a sterile 200 l pipette tip, the resultant monolayer was gently pulled in a straight line from the middle of each well. A 48-hour CO2 incubator was used to grow the cells after the media was withdrawn, the wells were washed in PBS (Sigma) solution, and BCAN substance dosages produced with DMEM medium containing 10% fetal bovine serum, 1% penicillin streptomycin, and 7.5% NaHCO3 were added to each well.. Images of scratch wound healing were captured at 0, 24, and 48 hours following scratch wound generation in HaCaT cells using an Olympus DP72 camera and an Olympus IX71 inverted microscope (both from Tokyo, Japan). Three replicates were used in the experiment.