Fluorescent PL accumulation in <i>Leishmania</i> parasites.

<p>Stationary promastigotes of <i>Leishmania</i> were incubated with the fluorescent PL analogues NBD-PS (A), NBD-PC (B), NBD-PE (C) or NBD-SM (D) for 30 min at 28°C. After washing and back-exchange with BSA, cell-associated fluorescence was measured by flow cytometry analysis. The grey histogram represents control cells transfected with the empty vector, the uncoloured histogram represents parasites expressing LABCG2^K/M and the dotted histogram represents non-labelled cells. (E) NBD-PS accumulation in <i>Leishmania</i> lines after a second transfection event. The reverted line (F), which was maintained for 3 months without drug pressure, was also included. The histograms correspond to a representative experiment from three independent experiments. (G) Outward transport of NBD-PS in <i>Leishmania</i> parasites. Stationary promastigotes of control (black circles) and LABCG2^K/M (black squares) <i>Leishmania</i> lines were incubated with the fluorescent analogue NBD-PS for 30 min at 28°C. After washing and back-exchange with BSA, cells were incubated for different time points in a free NBD-PS medium containing BSA and the fluorescence of the supernatant was measured by spectrofluorimetry. Results represent the means ± SD of four independent duplicated experiments. * <i>P</i><0.05 vs. control parasites.