Outward and inward transport of NBD-PS in Melan-A–specific CTLs. (A) Melan-A–specific CTLs were intracellularly loaded with NBD-PS, washed, and then incubated at 37°C in complete medium in the absence (▴) or presence (○) of Ag-loaded T2 cells at a CTL/T2 ratio of 3:1. Alternatively, CTLs were stimulated with ionomycin (▪). To trap NBD lipids appearing on the cell surface, incubation was performed in the presence of 1% HSA. Aliquots of cells were removed at various time points and analyzed by flow cytometry after addition of 10 mM dithionite to reduce the NBD moiety of analogues in the medium. Only PI^– CTLs were included for analysis. Relative NBD-PS fluorescence intensities at the start of the experiment were about 2400 arbitrary units. Shown are representative data of 3 independent experiments. (B) Ag-specific CTLs were stimulated with T2 cells (37°C for 45 minutes, ○) or 5 μM ionomycin (37°C for 15 minutes, ▪), or left untreated (▴), labeled with NBD-PS on ice, and incubated at 37°C to allow PS inward transfer. At times indicated, the fraction of internalized label was determined (in gated CD8⁺ PI^–) CTLs by flow cytometry using back-exchange to HSA and treatment with dithionite as described in “Materials and methods.” The relative fluorescence protected against dithionite is shown for one representative experiment of 3. In panels A and B the percentage of nonaccessible fluorescence was normalized to fluorescence at time point zero. The absolute values of nonaccessible fluorescence at time point zero ranged from 10% to 20%.