Cellsens rt software

Melanoma A375 and RPMI7951 cells were plated on Lab-Tek Chamber Slides (NUNC, Roskilde, Denmark) at the density of 4 × 10⁴ cells/mL and 8 × 10⁴ cells/mL, respectively. The next day, cells were exposed to serial dilutions of tested compound (L-KYN: 10⁻⁶, 10⁻³, 1 mM; KYNA: 10⁻⁶, 10⁻³, 5 mM, FICZ: 10⁻³, 1, 50 µM) or fresh cell culture medium (control, C) according to the experiment design described in detail above. After 24 h treatment, the effect of L-KYN, KYNA and FICZ on induction of cell death was analyzed after fluorescence staining with Hoechst 33342 and propidium iodide at a concentration of 0.2 μg/mL and 0.4 μg/mL, respectively (5 min at 37 °C). Cell images were captured with fluorescence microscopy (Olympus IX83 System Microscope; Olympus Optical Co., Ltd. and CellSens RT software, Olympus Optical Co., Ltd., Tokyo, Japan) at 10× magnification.

A375 cells cultured on LabTek Chamber Slides (Nunc, ThermoFisher Scientific, Roskilde, Denmark) were exposed to culture medium (control, C) or L-KYN 5 mM for 24 h in standard conditions according to the experiment design described in detail above. Then, cells were fixed with 3.7% paraformaldehyde, permeabilized with 0.2% Triton X-100, and treated with 5% bovine serum albumin (BSA, Sigma Aldrich, St. Louis, MO, USA). The cells were exposed to primary antibodies against cyclin D1 and CDK4 (1:100; Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C and then were incubated with secondary antibodies conjugated with fluorescein isothiocyanate (FITC) (1:100) (Sigma Aldrich, St. Louis, MO, USA) for 2 h at room temperature. Cell nuclei were labeled with cell permeable fluorescent DNA dye DraQ5 (Cell Signaling Technology, Danvers, MA, USA). Cell images were captured with fluorescence microscopy (automatic microscope Olympus IX83; Olympus Optical Co., Ltd., Tokyo, Japan, and CellSens RT software, Olympus Optical Co., Ltd., Tokyo, Japan) at 40× magnification.

Briefly, melanoma SK-MEL-3 cells were seeded on Lab-Tek Chamber Slide (Nunc, Roskilde, Denmark) at a density of 6 × 10⁴ cells/mL the day before the treatments as per the experimental design described above (KYN: 10⁻⁶, and 10⁻³, 1 mM; KYNA: 10⁻⁶, 10⁻³, and 5 mM; UVB: 20 mJ/cm²). After 24 h incubation, the effect of KYN and KYNA on induction of cell death was analyzed after fluorescence staining with Hoechst 33342 and propidium iodide as mentioned in detail previously [11 (link)].
Visualization was performed using fluorescence microscopy (Olympus IX83 System Microscope; Olympus Optical Co. Ltd. and CellSens RT software, Olympus Optical Co., Ltd., Tokyo, Japan) at 10× magnification.