A colorimetric assay for the measurement of D-glucose consumption by cultured cells.
A colorimetric method is described for measuring glucose consumption by tissue culture cells. This procedure, which utilizes the coupled activities of glucose oxidase and horseradish peroxidase, is insensitive to the spectral interferences caused by the phenol red and sera present in most tissue culture media. The spectral properties (absorbance maxima and apparent absorption coefficients) and stability of a large number of chromogenic horseradish peroxidase substrates were surveyed for their ability to perform in an assay for glucose in the presence of phenol red and sera components. One of these chromophores, the product of an oxidative couple between 4-aminoantipyrine and N-ethyl-N-sulfopropyl-m-toluidine, was subsequently used to develop a fixed time assay for glucose in media samples. The assay required only 10 microliters of media in a 1-ml assay volume; reproducibility studies showed variabilities of less than 5% in the assay of a single sample, and values obtained in glucose analyses correlated well with those obtained using commercially available test kits. The assay was used to study the rate of glucose consumption in two different cell types, bovine corneal endothelial cells and human diploid fibroblasts.
Blake
D A
DA
Department of Biochemistry, Meharry Medical College, Nashville, Tennessee 37208.
McLean
N V
NV
D000818:Animals; D002478:Cells, Cultured; D002863:Chromogenic Compounds; D005947:Glucose / Q000378:metabolism*; D005949:Glucose Oxidase; D006735:Horseradish Peroxidase; D006801:Humans; D013053:Spectrophotometry
D002863:Chromogenic Compounds; D005949:Glucose Oxidase; D006735:Horseradish Peroxidase; D005947:Glucose