A novel in vitro screening assay for trypanocidal activity using the fluorescent dye BCECF-AM.

Abstract

A cell viability assay, using fluorescence measurements has been developed for the screening of new compounds against African trypanosomes. 2',7'-Bis-(carboxyethyl)-5(6)-carboxyfluorescein-pentaacetoxymethyles ter (BCECF-AM), an esterase substrate, was used in the assay as a marker for cell viability. Fluorescence was quantified using an automated fluorescence scanner for multi-well plates. Trypanosoma brucei rhodesiense, T. congolense, T. evansi and T. equiperdum from continuously growing cultures were exposed to various concentrations of trypanocidal drugs for an incubation period of 72 h at 37 degrees C. Then BCECF-AM was added to the cell suspensions and after 60 minutes the fluorescence of the trypanosome suspension was measured using the Millipore Cytofluor 2300 fluorescence scanner, at 485 nm excitation and 530 nm emission wavelengths. Results of kinetic studies of the hydrolysis of the non-fluorescent BCECF-AM in trypanosomes showed that BCECF-AM is readily cleaved by non-specific esterases to a highly fluorescent product. Drug concentrations causing 50% inhibition of fluorescence (IC50-values) were measured fluorimetrically. Minimum inhibitory concentration (MIC) was determined microscopically.