Fluorescent microplate cell assay to measure uptake and metabolism of glucose in normal human lung fibroblasts.
This is the first report of a fluorimetric microplate assay to assess glucose uptake and metabolism in eukaryotic cells. The assay was carried out incubating normal human lung fibroblasts in the wells of microtiter trays with a fluorescent D-glucose derivative, 2-N-7-(nitrobenz-2-oxa-1,3-diazol-4-yl)amino-2-deoxy-D-glucose (2-NBDG). This dye could be incorporated by glucose transporting systems in living cells. Substrate uptake was determined by analysing the data obtained with a fluorescence microplate reader. Variables studied in the development of the assay included dye concentration and incubation period. We found that this cell assay is very sensitive, reproducible, provides fast results and graphical display of data. It requires small sample volumes and allows handling of a large number of samples simultaneously. Okadaic acid was used to assess this microplate assay in the field of cytotoxicity. This diarrhetic shellfish toxin is a tumour promoter and a specific inhibitor of protein phosphatases 1 and 2A. The exposition of cells to okadaic acid (0.1 nM-1 microM) at different time intervals causes a decrease in intracellular glucose (40-50% over controls). Results obtained with okadaic acid are the starting point to evaluate application of the method to routine toxicity probes.
Leira
F
F
ANFACO-CECOPESCA, Campus Universitario de Vigo, 36310 Vigo (Pontevedra), Spain.
Louzao
M C
MC
Vieites
J M
JM
Botana
L M
LM
Vieytes
M R
MR
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000378:metabolism*; D002273:Carcinogens / Q000633:toxicity; D002478:Cells, Cultured; D003847:Deoxyglucose / Q000031:analogs & derivatives* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000633:toxicity; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D005456:Fluorescent Dyes / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D008168:Lung / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D008832:Microchemistry / Q000379:methods*; D019319:Okadaic Acid / Q000633:toxicity; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D002273:Carcinogens; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D019319:Okadaic Acid; D003847:Deoxyglucose; D009327:4-Chloro-7-nitrobenzofurazan; D005947:Glucose; C098340:2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose