Development of a semi-automated colorimetric assay for screening anti-leishmanial agents.
MTS or {3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl}-2H-tetrazolium, inner salt) is converted into soluble formazan by mitochondrial dehydrogenase of viable cells, thus serving as an indicator of cell viability. Accordingly, a MTS-based assay was developed to evaluate anti-leishmanial activity in Leishmania promastigotes from strains responsible for visceral, cutaneous or mucocutaneous leishmaniasis. The assay was initially optimized for the appropriate wavelength (490 nm), culture medium (M-199), incubation time (3 h) and temperature (37 degrees C). Increasing absorbance with increasing cell density confirmed linearity of the assay that was maintained up to 2.5 x 10(6) cells/200 microl. The growth kinetics of six L. donovani strains and six non-L. donovani strains consistently indicated higher absorbances in the L. donovani strains highlighting the importance of strain-specific customization of the MTS assay. The IC(50) values (i.e., the concentration at which 50% of growth was inhibited) of amphotericin B, miltefosine and pentamidine isethionate obtained by the MTS assay corroborated with previously published data. Taken together, the MTS assay thus permits a simple, reproducible and reliable semi-automated method for evaluating cell viability, effective for drug-screening and growth kinetic studies.
Ganguly
Sudipto
S
Department of Pharmacology, Institute of Post Graduate Medical Education and Research, 244B Acharya J.C. Bose Road, Kolkata-700 020, West Bengal, India.
Bandyopadhyay
Samiran
S
Sarkar
Arup
A
Chatterjee
Mitali
M
D000666:Amphotericin B / Q000494:pharmacology; D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D003124:Colorimetry / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry*; D020128:Inhibitory Concentration 50; D007700:Kinetics; D007891:Leishmania / Q000187:drug effects* / Q000302:isolation & purification; D007893:Leishmania donovani / Q000187:drug effects / Q000302:isolation & purification; D007898:Leishmaniasis, Visceral / Q000469:parasitology; D010419:Pentamidine / Q000494:pharmacology; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000494:pharmacology; D013778:Tetrazolium Salts / Q000737:chemistry* / Q000378:metabolism; D013844:Thiazoles / Q000737:chemistry* / Q000378:metabolism
D000981:Antiprotozoal Agents; D007202:Indicators and Reagents; D013778:Tetrazolium Salts; D013844:Thiazoles; D010767:Phosphorylcholine; C070380:3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; C039128:miltefosine; D010419:Pentamidine; D000666:Amphotericin B