Solution 8

The potential of the mitochondrial membrane was evaluated using the image cytometry technique, which allows demarcation of the percentage of cells with a polarized or depolarized mitochondrial membrane. The analysis is based on a fluorescent cationic dye JC-1 accumulating inside the mitochondria with a high transmembrane potential. At high concentrations, JC-1 aggregates and shows red fluorescence. In turn, JC-1 localizes in the cytoplasm in cells with a low mitochondrial potential and then shows green fluorescence. The tested melanoma cells were stained with JC-1 after the treatment by adding 12.5 µL of Solution 7 (200 µg/mL JC-1) (ChemoMetec, Lillerød, Denmark) to cell suspensions in PBS (1.0 × 10⁶ cells/mL). Then, the samples were incubated for 30 min at 37 °C, centrifuged and washed twice with PBS. Immediately before the analysis, the cells were suspended with 0.25 mL of Solution 8 (1 µg/mL DAPI in PBS) (ChemoMetec, Lillerød, Denmark). The analysis was made according to the Mitochondrial Potential Assay protocol using NucleoCounter^® NC-3000™.

Meloxicam was obtained from Boehringer Ingelheim (Budapest, Hungary). Fibroblasts Growth Medium, amphotericin B solution (250 µg/mL), penicillin, phosphate-buffered saline (PBS) and L-3,4-dihydroxyphenylalanine (L-DOPA) were purchased from Sigma Aldrich Inc. (St. Louis, MO, USA). Neomycin sulfate was acquired from Amara (Kraków, Poland). An M-254 growth medium and a human melanocyte growth supplement-2 (HMGS-2) were obtained from Cascade Biologics (Portland, OR, USA). Trypsin/EDTA was obtained from Cytogen (Zgierz, Poland). Via-1-Cassettes™ (acridine orange and DAPI fluorophores), NC-slides A2 and A8, as well as Solution 3 (1 µg/mL DAPI, 0.1% triton X-100 in PBS), Solution 5 (VB-48™ PI AO), Solution 7 (200 µg/mL JC-1) and Solution 8 (1 µg/mL DAPI in PBS) were purchased from ChemoMetec (Lillerød, Denmark). H₂DCFDA reagent was acquired from Thermo Fisher Scientific Inc. (Waltham, MA, USA). WST-1 cell proliferation reagent was obtained from Roche GmbH (Mannheim, Germany). Other chemicals were obtained from POCH S.A. (Gliwice, Poland).