Msd 5972

Total and free intracellular zinc content in the explant human melanoma cultures, human melanoma cell line Bowes, and normal human melanocytes HEM were determined as described before [47 (link)]. The trypsinized and rinsed cells were dissolved in 0.35 mL 0.8% nitric acid and assayed for zinc with the inductively coupled plasma emission spectrometer MSD 5972 (Agilent Technologies, Waldbronn, Germany). Prior to analysis, aliquots of the cell samples were assayed for protein content using a BCA assay (Bicinchoninic acid kit for protein determination, Sigma-Aldrich, Prague, Czech Republic). Changes in total intracellular zinc content were expressed as µg of zinc/mg of protein.
Free intracellular zinc levels in the assayed cells were determined fluorimetrically. The cells grown in black-bottom 96-well plates were incubated with Newport Green diacetate (5 μmol in PBS, dark, 30 min at 37 °C). Fluorescence intensity was determined by the multiplate reader TECAN SpectraFluor Plus (TECAN Austria GmbH, Grödig, Austria). The results in relative light units were obtained from the raw data minus reagent blank, with changes expressed as a percentage of controls.

The trypsined and rinsed melanoma cells were harvested (approximate 0.2 g of wet mass), placed into quartz vessels, and digested with 3.5 mL 60% nitric acid. Thereafter, sealed vessels were microwave oven heated, rinsed in ultrapure water, and aliquots diluted 1:10 prior to zinc concentration analysis with the inductively coupled plasma emission spectrometer MSD 5972 (Agilent Technologies, Waldbronn, Germany) as based on [35 (link),36 (link)]. Prior to analysis, aliquots of the cell samples were assayed for protein content using a BCA assay (Bicinchoninic acid kit for protein determination, Sigma-Aldrich, Prague, Czech Republic). Changes in total intracellular zinc content were expressed as µg of zinc/mg of protein.
Free intracellular zinc levels in the assayed melanoma cells were determined microfluorimetrically based on the published protocol [37 (link)]. The cells grown in black-bottom 96-well plates were incubated with Newport Green diacetate (5 μM in PBS, dark, 30 min at 37 °C). Fluorescence intensity was determined by the multiplate reader TECAN SpectraFluor Plus (TECAN Austria GmbH, Grödig, Austria). The results in relative light units were obtained from the raw data minus reagent blank, with changes expressed as a percentage of controls.