Flow cytometric determination of intracellular non-protein thiols in Leishmania promastigotes using 5-chloromethyl fluorescein diacetate.

Leishmania parasites lack catalase and therefore, their anti-oxidant system hinges primarily upon non protein thiols; accordingly, depletion of thiols could potentially serve as an effective drug target. We have developed a flow cytometry based assay using 5-chloromethyl fluorescein diacetate based upon its selective staining of non protein thiols. Its specificity was confirmed using buthionine sulphoximine (a gamma glutamyl cysteine synthetase inhibitor), diamide (an oxidizing agent of intracellular thiols) and N-ethylmaleimide (a covalent modifier of cysteine residues) as evidenced by reduction in fluorescence; furthermore, restoration of fluorescence by N-acetyl cysteine corroborated specificity of 5-chloromethyl fluorescein diacetate to measure non protein thiols. Differences in basal level of thiols in antimony sensitive and antimony resistant Leishmania field isolates were detected. The depletion of non protein thiols by conventional antileishmanial drugs e.g. antimony and miltefosine was demonstrated. Furthermore, fluorescence was unaffected by depletion of ATP in majority of the strains studied, indicating that 5-chloromethyl fluorescein diacetate is not a substrate for the pump operative in most Leishmania donovani strains. Taken together, measurement of 5-chloromethyl fluorescein diacetate fluorescence is an effective method for monitoring non protein thiols in Leishmania promastigotes.