Enzyme activities of six different dehydrogenases in Ehrlich ascites cells measured by flow cytometry.
Flow cytometric measurements of the activities of lactate dehydrogenase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, glycerol-3-phosphate dehydrogenase, succinate dehydrogenase and glutamate dehydrogenase in single Ehrlich ascites tumour cells are described using a tetrazolium salt/fluorescent formazan reaction. Applying cyano-ditolyl-tetrazolium chloride (CTC) as redox dye indicating enzyme reaction, and DAPI as a fluorochrome for nuclear DNA staining, the bivariate flow cytometric assay of enzyme activity and cell cycle analysis was established. Furthermore, adopting the calibration procedure reported formerly, consisting of biochemical determination and flow cytometry of the same sample performed parallelly, the enzyme activities were expressed in biochemical units. The dehydrogenase activities found in Ehrlich ascites cells were 97.5 fmol H2 per average positive cell during 5 min for lactate dehydrogenase, 69.0, 10.6, 25.3, 29.7, and 19.0 fmol H2 per average positive cell during 20 min for glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, glycerol-3-phosphate dehydrogenase, succinate dehydrogenase and glutamate dehydrogenase, respectively. This quantitative procedure can offer an alternative analytic tool for enzyme cytology.
Huang
C J
CJ
Institut für Strahlenbiologie der Universität Münster, Germany.
Severin
E
E
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology* / Q000473:pathology; D005434:Flow Cytometry; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D005993:Glycerolphosphate Dehydrogenase / Q000378:metabolism; D006651:Histocytochemistry; D007211:Indoles / Q000737:chemistry; D007521:Isocitrate Dehydrogenase / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D051379:Mice; D010088:Oxidoreductases / Q000378:metabolism*; D013385:Succinate Dehydrogenase / Q000378:metabolism; D013778:Tetrazolium Salts / Q000737:chemistry
D007211:Indoles; D013778:Tetrazolium Salts; C075453:5-cyano-2,3-ditolyltetrazolium; C007293:DAPI; D010088:Oxidoreductases; D005993:Glycerolphosphate Dehydrogenase; D007770:L-Lactate Dehydrogenase; D007521:Isocitrate Dehydrogenase; D005954:Glucosephosphate Dehydrogenase; D013385:Succinate Dehydrogenase; D005969:Glutamate Dehydrogenase