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

Sarkar, Avijit ; Mandal, Goutam ; Singh, Neeloo ; Sundar, Shyam ; Chatterjee, Mitali (2009) Flow cytometric determination of intracellular non-protein thiols in Leishmania promastigotes using 5-chloromethyl fluorescein diacetate Experimental Parasitology, 122 (4). pp. 299-305. ISSN 0014-4894

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Official URL: http://www.sciencedirect.com/science/article/pii/S...

Related URL: http://dx.doi.org/10.1016/j.exppara.2009.04.012

Abstract

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 γ-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 anti-leishmanial 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.

Item Type:Article
Source:Copyright of this article belongs to Elsevier Science.
Keywords:CMFDA; Thiols; Leishmaniasis; Antileishmanial; Promastigotes; Antimony Resistance
ID Code:94525
Deposited On:17 Sep 2012 10:52
Last Modified:17 Sep 2012 10:52

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