Flow cytometric analysis of drug efflux.

The accumulation of 5(6)-carboxy-2′-7′-dichlorofluorescein (CDCF; Sigma-Aldrich) was evaluated upon incubation of macrophages (2 × 10⁵ cells/measurement) with 160 μM CDCF in RPMI 1640 without phenol red for 1 h at 4°C. When experiments were performed with Leishmania-infected macrophages, the cells were first seeded in a T75 culture flask in RPMI 1640 medium and infected with ex vivo amastigotes for 24 h. After incubation, the cells were washed twice with cold medium and an aliquot was taken to quantify CDCF uptake by flow cytometry in the fluorescein isothiocyanate channel (FACSCalibur flow cytometer; Becton, Dickinson). Dye retention in macrophages was evaluated after 2 h of incubation at 37°C in the absence (negative control) or presence (positive control) of probenecid at 4 mM alongside the DNDi compounds and Sb^III at 10 μM. To assess cell viability, 7-amino actinomycin D (7-AAD; Biosciences) was added. Data analysis and determination of the mean fluorescence intensity were carried out with Flow Jo X software.