A COLORIMETRIC ASSAY FOR THE MEASUREMENT OF D-GLUCOSE CONSUMPTION BY CULTURED-CELLS
A COLORIMETRIC ASSAY FOR THE MEASUREMENT OF D-GLUCOSE CONSUMPTION BY CULTURED-CELLS
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DOI：
10.1016/0003-2697(89)90031-6
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发表时间：
1989-02-15
影响因子：
2.9
通讯作者：
MCLEAN, NV
中科院分区：
文献类型：
--
作者：
BLAKE, DA;MCLEAN, NV
描述了一种测量组织培养细胞葡萄糖消耗的比色法。该方法利用葡萄糖氧化酶和辣根过氧化物酶的偶联活性，对大多数组织培养基中存在的酚红和血清引起的光谱干扰不敏感。考察了大量辣根过氧化物酶显色底物的光谱性质(最大吸光度和表观吸收系数)和稳定性，考察了它们在苯酚、红和血清成分存在下测定葡萄糖的能力。其中一个发色团是4-氨基安替比林和N-乙基-N-磺基丙基-间甲苯胺氧化偶联的产物，随后被用来建立测定介质样品中葡萄糖的固定时间分析方法。在1毫升的测定体积中，该测定只需要10µl的介质；重复性研究表明，单个样品的测定的变异小于5%，并且葡萄糖分析中获得的值与使用商业可获得的检测试剂盒获得的值具有很好的相关性。该方法用于研究两种不同类型的细胞--牛角膜内皮细胞和人二倍体成纤维细胞的葡萄糖消耗率。
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 .mu.l 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.