TY - JOUR
T1 - Development of a semi-automated colorimetric assay for screening anti-leishmanial agents
AU - Ganguly, Sudipto
AU - Bandyopadhyay, Samiran
AU - Sarkar, Arup
AU - Chatterjee, Mitali
N1 - Funding Information:
This work was supported by the University Grants' Commission, Council of Scientific and Industrial Research, Govt. of India and Life Sciences Research Board, Ministry of Defence, Govt. of India. We thank Dr. Vanessa Yardley, London School of Tropical Medicine and Hygiene, UK and Dr. Neeloo Singh, Central Drugs Research Institute, CDRI, Lucknow, for having kindly provided us parasite strains. The L. donovani species specific monoclonal antibody was a kind gift from Prof. C.L. Jaffe, Dept. of Parasitology, Hadassah Medical School, Jerusalem, Israel. Mr. Sudipto Ganguly is a recipient of a Junior Research Fellowship from the University Grants' Commission and Dr. Samiran Bandyopadhyay is a recipient of a Senior Research Fellowship from Indian Council of Medical Research.
PY - 2006/7
Y1 - 2006/7
N2 - 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 °C). Increasing absorbance with increasing cell density confirmed linearity of the assay that was maintained up to 2.5 × 106 cells/200 μl. 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 IC50 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.
AB - 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 °C). Increasing absorbance with increasing cell density confirmed linearity of the assay that was maintained up to 2.5 × 106 cells/200 μl. 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 IC50 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.
KW - Anti-leishmanial activity
KW - Cell viability
KW - Drug screening
KW - Leishmaniasis
KW - MTS
KW - Promastigotes
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U2 - 10.1016/j.mimet.2005.10.011
DO - 10.1016/j.mimet.2005.10.011
M3 - Article
C2 - 16316700
AN - SCOPUS:33646785770
SN - 0167-7012
VL - 66
SP - 79
EP - 86
JO - Journal of Microbiological Methods
JF - Journal of Microbiological Methods
IS - 1
ER -