Determination of pyridine nucleotide contents of cell monolayers by bioluminescence.
In this paper, we describe methods to assay specifically the NAD+, the NADP+, the NADH and the NADPH contents of cell monolayers. After an acid or an alkaline extraction, respectively for the oxidized or the reduced pyridine nucleotides, each type of nucleotide can be separately quantified with the use first, of specific reducing enzymes (lactate or glucose-6-phosphate dehydrogenases), followed by a bioluminescence enzymatic reaction [NADP(H)-FMN oxidoreductase and luciferase]. The assays hence developed are sensitive, reliable and specific. An application of the method with pulmonary alveolar macrophage monolayers is also described.
Lane
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Département de biologie, Faculté des sciences, Université de Sherbrooke, Québec, Canada.
Nadeau
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D000818:Animals; D008156:Luciferases / Q000032:analysis; D008163:Luminescent Measurements; D008264:Macrophages / Q000032:analysis*; D008297:Male; D008722:Methods; D009243:NAD / Q000032:analysis*; D009249:NADP / Q000032:analysis*; D010088:Oxidoreductases / Q000032:analysis; D051381:Rats; D012015:Reference Standards
D009243:NAD; D009249:NADP; D010088:Oxidoreductases; D008156:Luciferases