In situ tracing of ROS formation in MEL cells. The various MEL clones were treated with the indicated concentration of H₂O₂ after loading with the nonfluorescent carboxy-2′,7′-di-Cl-fluorescein (CDCF) permeant analog (CDCFDA). The latter is converted intracellularly into the fluorescent analog by reacting with ROS in a metal-dependent fashion. Cells were preincubated for 10 minutes with FAS (20 μmol/L) (A, bottom) or buffered saline (B, top) and washed. Cell fluorescence was analyzed by FACS at different times after addition of H₂O₂. Data (n = 4 experiments run in triplicate samples) are given in terms of mean rates of fluorescence change with time (AFU/min = arbitrary fluorescence units/min) with SEM of less than 8% the indicated points in the graph. The increment in the fluorescence intensity ▵ attributable to FAS (▵ = A to B) (in AFU/min) was at 0, 10, and 20 μmol/L H₂O₂ respectively for wt: 0.31, 0.85, and 1.1; for cl-16: 0.45, 0.78, and 0.83; for cl-12: 0.18, 0.12, and 0.32; and for cl-6: 0.11, 0.10, and 0.10. ANOVA paired analysis of the n = 4 experiments showed statistically significant differences (P < .05) between data points at given H₂O₂ concentrations when cl-6 or cl-12 were compared with either cl-16 or wt but not when they were compared with each other.