Rapid fluorescent assay for screening drugs on Leishmania amastigotes

A rapid fluorescent viability assay employing alamarBlue was optimized for use with Leishmania axenic amastigotes, the stage of the parasite responsible for disease pathology. The activity of two protein kinase inhibitors, Staurosporine and H-89, as well as Amphotericin B, on promastigotes and amastigotes of Leishmania donovani and Leishmania tropica was compared. Both protein kinase inhibitors inhibited promastigote growth at lower concentrations than amastigotes, while the GI50 for Amphotericin B on both stages was similar. This assay only requires a limited number of axenic amastigotes (50,000 cells/well) and can be used to rapidly screen large chemical or natural product libraries for activity against amastigotes.