The use of a water-soluble formazan complex to quantitate the cell number and mitochondrial function ofLeishmania major promastigotes

Abstract

One of the methods to quantitate Leishmania major promastigotes (LmP) has been to utilize the formation of a formazan dye, which in turn is produced via conversion of an artificial substrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The method has one major drawback in that the formazan complex precipitates inside the parasites and has to be extracted by denaturants before measurements can be performed. By using a new synthetic substrate, 3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS), the extraction procedure is eliminated as the formazan-like dye is released spontaneously into the medium, making it possible to perform several measurements on the same parasite culture without disturbing or killing the parasites. The measurements were shown to reflect the numbers of parasites as confirmed via comparative experiments using radioactive thymidine uptake and cell counting, respectively. The method is simple, fast, and highly reproducible and is suitable for drug screening, identification of drug-resistant isolates, and growth-kinetics studies. It is therefore contemplated that the MTS method will be a general and useful technique in this field of parasitology.

Hydrosoluble formazan XTT: its application to natural products drug discovery for Leishmania

“…The data presented in Fig. 2 also confirm the reliability of the MTS-based methodology as previously shown by Berg et al (1994).…”

“…Natural products have been a rich source of compounds with antileishmanial activity (Chan-Bacab and Pena-Rodriguez, 2001), revealing the need for an efficient and quantitative method for screening large numbers of extracts and compounds for anti-parasitic activity. Incorporation of labeled [ 3 H]thymidine into the DNA of the multiplying parasite is a reliable assay (Berg et al, 1994), but the use and disposal of radioactive isotopes can be inconvenient or impossible, especially in laboratories based in unindustrialized countries. A colorimetric method based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by the mitochondrial dehydrogenase of the parasite is widely used for Leishmania growth and shows a high correlation with the radioactive method and with a technique that employs the water soluble formazan, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS) (Berg et al, 1994).…”

“…Incorporation of labeled [ 3 H]thymidine into the DNA of the multiplying parasite is a reliable assay (Berg et al, 1994), but the use and disposal of radioactive isotopes can be inconvenient or impossible, especially in laboratories based in unindustrialized countries. A colorimetric method based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by the mitochondrial dehydrogenase of the parasite is widely used for Leishmania growth and shows a high correlation with the radioactive method and with a technique that employs the water soluble formazan, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS) (Berg et al, 1994). However, the formazan product derived from the reduction of MTT is insoluble in aqueous media, requiring aspiration of the media and the addition of organic solvents in order to read optical density values (OD).…”

“…For measuring the proliferation of Leishmania promastigotes, Berg et al (1994) had previously shown a correlation between [ 3 H]thymidine uptake and the reduction of MTT and MTS. Accordingly, we compared the ability of L. mexicana to reduce MTT, MTS and XTT since these substrates are all enzymatically reduced by mitochondrial dehydrogenase (Buttke et al, 1999).…”

“…Accordingly, we compared the ability of L. mexicana to reduce MTT, MTS and XTT since these substrates are all enzymatically reduced by mitochondrial dehydrogenase (Buttke et al, 1999). The CellTiter 96R aqueous kit, employed for the MTS-based assay (Berg et al, 1994), was also used in the present study (Technical Bulletin #169,Promega). In this experiment, three identical 96-well plates with increasing number of L. mexicana were seeded in triplicate and were exposed to MTT, XTT or MTS.…”