Fig. 3. Decreased melanin contents in B16 melanoma cells treated with H₂O₂. (A) Cells challenged with H₂O₂ (0.5 mM, 20 minutes) were allowed to recover for the times shown. After harvesting with trypsin, the cells were solubilized in 1% Igepal CA-630 and centrifuged at 20,000 g, for 30 minutes. Protein concentration was determined in the supernatant, and total melanin was measured by KOH hydrolysis of the insoluble pellet. Results are the mean±s.d. of three independent experiments. (B) Growth kinetics of H₂O₂-treated cells. 4x10⁴ cells were seeded in six-well plates, in sets of triplicate wells, and incubated for 24 hours in the complete medium described in Materials and Methods. Then the cells were challenged with H₂O₂ (0.5 mM, 20 minutes) or with Hank’s solution, and further incubated in complete medium from 1 to 24 hours. After trypsinization, the number of viable cells was determined by Trypan Blue exclusion. Open symbols, H₂O₂-treated cells; closed symbols, control cells. Results are shown as mean ± s.d. of triplicate wells.