The Tetrazolium-Fomazan System: Design and Histochemistry

Seidler, E

doi:10.1016/s0079-6336(11)80060-4

Cited by 91 publications

(53 citation statements)

References 131 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The tetrazolium salt XTT was converted by Aspergillus species only in the presence of an electron-coupling agent, as was found with yeasts (14). As opposed to mammalian cells (12) but similar to yeasts (15), among the two electron-coupling agents tested menadione was more potent than PMS.…”

mentioning

confidence: 60%

See 1 more Smart Citation

Colorimetric Assay for Antifungal Susceptibility Testing of Aspergillus Species

Meletiadis¹,

et al. 2001

View full text Add to dashboard Cite

A colorimetric assay for antifungal susceptibility testing of Aspergillus species (Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus nidulans, and Aspergillus ustus) is described based on the reduction of the tetrazolium salt 2,3-bis(2-methoxy-4-nitro-5-[(sulphenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) in the presence of menadione as an electron-coupling agent. The combination of 200 g of XTT/ml with 25 M menadione resulted in a high production of formazan within 2 h of exposure, allowing the detection of hyphae formed by low inocula of 10 2 CFU/ml after 24 h of incubation. Under these settings, the formazan production correlated linearly with the fungal biomass and less-variable concentration effect curves for amphotericin B and itraconazole were obtained.Tetrazolium salts are heterocyclic organic compounds that substitute the natural final acceptor (oxygen) in the biological redox process and are reduced to formazan derivatives by receiving electrons enzymically from substances of the hydrogen transport system or nonenzymically from artificial electron transporters (phenazine methosulfate and menadione) which enhance the reaction. Tetrazolium salts can penetrate rapidly into intact cells and directly into subcellular membranes with dehydrogenase activity, where they are converted to colored formazan derivatives (1,14). Therefore, they were used as indicators of reducing systems. The tetrazolium salt MTT has been used for antifungal susceptibility testing of various yeasts and filamentous fungi, and testing was in agreement with the corresponding standard methods recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (2, 7). The disadvantage of MTT, however, is that the process includes the solubilization of formazan derivatives. As an alternative, a new tetrazolium salt, 2,3-bis(2-methoxy-4-nitro-5-[(sulphenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) (10), has been employed for antifungal susceptibility testing of yeasts and resulted in clear-cut endpoints for various antifungal agents (4, 15). XTT is converted into a water-soluble formazan, thereby avoiding the additional steps for the solubilization of formazan derivatives (8, 12), but needs the presence of an electron-coupling agent. The nature and the concentration of this agent are critical in order to obtain a good correlation between the formazan production and the number of viable fungi and less-variable concentration effect curves (1, 15).We developed a colorimetric assay for the quantification of fungal growth of five different Aspergillus species based on the tetrazolium salt XTT by standardizing various factors that influence XTT conversion. This assay was also tested in the presence of antifungal drugs in order to ascertain its potential for antifungal susceptibility testing of filamentous fungi.Isolates. Two clinical isolates of Aspergillus fumigatus [AZN5161 (S) and AZN 5241 (R)] and one each of Aspergillus flavus (AZN 510), Aspergillus terreus (AZN 7320), Aspergillus nidulans (AZN ...

show abstract

mentioning

confidence: 60%

“…Tetrazolium salts can penetrate rapidly into intact cells and directly into subcellular membranes with dehydrogenase activity, where they are converted to colored formazan derivatives (1,14). Therefore, they were used as indicators of reducing systems.…”

mentioning

confidence: 99%

Colorimetric Assay for Antifungal Susceptibility Testing of Aspergillus Species

Meletiadis¹,

et al. 2001

View full text Add to dashboard Cite

show abstract

“…For localization of dehydrogenase activity at the LM level, tetranitro BT or nitro BT, and at the EM level, BPST are recommended for at least two reasons. The first is that tetranitro BT, nitro BT, and BPST are reduced easily and give a high spatial resolution because formazan crystals are fine (Van Noorden and Butcher 1984;Seidler 1991). BPST is recommended for use at the EM level.…”

Section: Chemical Backgrounds Of Oxygen Insensitivitymentioning

confidence: 99%

“…It has always been assumed that an unknown factor is involved in NT reduction that is increased or decreased in cancer cells in comparison with normal cells. Because tetrazolium salts are reduced in steps of one electron at a time (Raap 1983;Seidler 1991) to produce unstable intermediates, tetrazolinyl radicals (·NT; Seidler and Van Noorden 1994), it was always believed that oxygen radicals (O 2˙ Ϫ ) were involved. ·NT can donate its unpared electron to O 2 to form O 2˙ Ϫ and NT.…”

Section: Chemical Backgrounds Of Oxygen Insensitivitymentioning

confidence: 99%

Oxygen Insensitivity of the Histochemical Assay of Glucose-6-Phosphate Dehydrogenase Activity for the Discrimination Between Nonmalignant and Malignant Cells

Driel

Noorden

1999

J Histochem Cytochem.

View full text Add to dashboard Cite

SUMMARYWe review here the oxygen insensitivity of the histochemical assay of glucose-6-phosphate dehydrogenase (G6PDH) activity to detect cancer cells. This inexpensive and rapid assay can be performed within half an hour. Discrimination between cancerous and noncancerous cells is based on a combination of elevated G6PDH activity, decreased superoxide dismutase (SOD) activity, and decreased lipid peroxidation in cancer cells. The test discriminates between adenomas and carcinomas of the colon with a certainty of Ͼ80% and has a high prognostic value for survival of colon cancer patients. Pancreatitis and pancreatic cancer are discriminated with a certainty of 100%. Therefore, the test can be applied by pathologists to provide additional information in difficult cases of diagnosis of cancer and for prognosis.

show abstract

“…Direct measurement is possible providing that the reducibility of the tetrazolium salts is higher than that of CTC (unpublished results) and NT (Seidler 1991), the tetrazolium salt used by Macha et al (1975) in their coupled reaction procedure. The lesser reducibility of CTC in comparison to NBT favors its enzymatic reduction, thus preventing nonspecific reactions (Seidler 1991). We therefore estimate that not more than 5% of the total amount of CTC formazan is produced nonenzymatically in our experiments.…”

Section: Discussionmentioning

confidence: 99%

Flow Cytometric Assay of Cytochemically Demonstrated NAD(P)H Oxidoreductase (Diaphorase) Activities

Severin

Seidler²

1998

J Histochem Cytochem.

Self Cite

View full text Add to dashboard Cite

The tetrazolium salt 5-cyano-2,3-di- p-toluyl-tetrazolium chloride (CTC), yielding a fluorescent formazan on reduction, was used to measure NAD(P)H oxidoreductase activity. In this study, optimal conditions for the flow cytometric technique were determined empirically with tissue culture cell lines and mouse Ehrlich ascites cells. Applying a coupled reaction procedure, NADH and NADPH as substrates of the oxidoreductases to be measured are generated endogenously by lactate or glucose-6-phosphate dehydrogenase, respectively. The results were evaluated by combining spectrophotometry and flow cytometry. We obtained integral activities for each group of NADH and NADPH oxidoreductases. Furthermore, by counterstaining the DNA with DAPI, followed by bivariate analysis of flow cytometric data, our assay gives a detailed distribution of enzyme activities of all cells, even in subgroups present in heterogeneous cell populations. Therefore, this protocol permits the study of NAD(P)H oxidoreductase activities in ex vivo tumor samples in which mixed cellular populations may be present.

show abstract

The Tetrazolium-Fomazan System: Design and Histochemistry

Cited by 91 publications

References 131 publications

Colorimetric Assay for Antifungal Susceptibility Testing of Aspergillus Species

Colorimetric Assay for Antifungal Susceptibility Testing of Aspergillus Species

Oxygen Insensitivity of the Histochemical Assay of Glucose-6-Phosphate Dehydrogenase Activity for the Discrimination Between Nonmalignant and Malignant Cells

Flow Cytometric Assay of Cytochemically Demonstrated NAD(P)H Oxidoreductase (Diaphorase) Activities

Contact Info

Product

Resources

About