, 2004). Briefly, 3 × 106 B16F10 cells were seeded in 96-well plates and incubated with G8 and G12 for 24 h. DNA amount was quantified spectrophotometrically (ε260 = 20 mg−1 cm−1) ( Cavaluzzi and Borer, 2004). Caspase-3 activity was monitored by the production
of fluorescent AMC from DEVD-AMC fluorogenic substrate for caspase-3 and related cysteine proteases. Briefly, B16F10 cells were seeded in a six-well plate (1 × 106 cells/well) and treated with 50 μM of G8 and G12 for a time period ranging from 15 min to 24 h. Cleavage of the fluorogenic substrate was detected spectrofluorimetrically (Perkin Elmer LS55, Boston, MA, USA) after 2 h of incubation at 37 °C, using excitation and emission wavelengths of 380 and 460 nm, respectively (Zuse et al., 2007). The result was expressed in arbitrary units of fluorescence, considering the activity of the control as one unit. The possible selleck chemical effect of G8 and G12 in disturbing mitochondrial membrane potential was evaluated using the fluorescent probe JC-1. B16F10 cells were seeded in 12-well plates (5 × 105 cells/well) and incubated with 50 μM of G8 and G12 for 15 min at 37 °C. At the end GKT137831 in vivo of the incubation time, cells were stained with 10 μg/ml JC-1 for 20 min at 37 °C.
The evaluation was performed by the visualization of the cells under a fluorescence microscope (Nikon Eclipse TS100 inverted microscope, Nikon Instruments Inc., Melville, NY, USA) and by the quantification of green and red fluorescences spectrofluorimetrically (Perkin Elmer LS55, Boston, MA, USA). The excitation wavelength for JC-1 is 488 nm, and the red and green emission fluorescence were detected at 590 and 527 nm, respectively (Cossarizza et al., 1993). The red/green fluorescence ratio for each sample was calculated and normalized as a percentage of the untreated control (100%). The changes in mitochondrial potential were indicated as a decrease in the red and green fluorescence intensity ratio. FCCP (1 μM), a chemical electron transport uncoupler, was used as a positive control. The
production of intracellular reactive species was evaluated using the DCFH2-DA probe. B16F10 Cyclooxygenase (COX) cells were seeded in 24-well plates (3 × 105 cells/well) and incubated for 3 h with 50 μM of G8 and G12. At the end of the incubation time, cells were stained with 10 μM DCFH2-DA for 30 min at 37 °C. The evaluation was performed by the visualization of cells under fluorescence microscope and by the quantification of green fluorescence in the spectrofluorimeter (Perkin Elmer LS55, Boston, MA, USA) after the cells’ removal from the culture plate, using wavelengths of excitation/emission of 480/520 nm, respectively (Sauer et al., 2003). An analytical curve was performed using a standard DCF solution to analyze the results, which were subsequently normalized as a percentage of the untreated control (100%). B16F10 cells were seeded in six-well plates (3 × 106 cells/well) and incubated with 50 μM of the gallates for 3 h at 37 °C.