Flow Cytometric Assay of Cytochemically Demonstrated NAD(P)H Oxidoreductase (Diaphorase) Activities
| Autor: | Eberhard Seidler, Erhard Severin |
|---|---|
| Rok vydání: | 1998 |
| Předmět: |
0301 basic medicine
Histology Dehydrogenase Glucosephosphate Dehydrogenase Flow cytometry Mice 03 medical and health sciences chemistry.chemical_compound Oxidoreductase Diaphorase Tumor Cells Cultured medicine Animals Endothelium DAPI chemistry.chemical_classification L-Lactate Dehydrogenase 030102 biochemistry & molecular biology biology medicine.diagnostic_test NADPH Dehydrogenase DNA Flow Cytometry Molecular biology Enzyme assay 030104 developmental biology chemistry Biochemistry biology.protein NAD+ kinase Anatomy Formazan |
| Zdroj: | Journal of Histochemistry & Cytochemistry. 46:761-765 |
| ISSN: | 1551-5044 0022-1554 |
| DOI: | 10.1177/002215549804600609 |
| Popis: | SUMMARY 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. (J Histochem Cytochem 46:761‐765, 1998) |
| Databáze: | OpenAIRE |
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