Gómez-Lechón M J, Ponsoda X, Castell J V
Unidad de Hepatología Experimental, Hospital Universitario La Fe, Valencia, Spain.
Anal Biochem. 1996 May 1;236(2):296-301. doi: 10.1006/abio.1996.0170.
This study describes a rapid, sensitive, and automated spectrophotometric enzymatic microassay that measures the intracellular glycogen of primary cultured hepatocytes and other cultured cells in 96-well plates and can be adapted for other samples that are transferred to these plates. The procedure involves in situ disruption of cells, followed by hydrolysis of glycogen into glucosyl units by fungal glucoamylase (exo-1.4-alpha-glucosidase, EC 3.2.1.3), and glucose determination with the glucose oxidase colorimetric method. The color intensity can be measured in conventional ELISA readers, and the data can be fed to an on-line computer for rapid processing. The advantages of this method are its simplicity and automation, the reduction in sample handling, and the small number of cells required compared to other conventional methods.
本研究描述了一种快速、灵敏且自动化的分光光度酶微测定法，该方法可测量原代培养肝细胞和其他培养细胞在96孔板中的细胞内糖原含量，并且可适用于转移至这些孔板的其他样品。该程序包括原位破坏细胞，随后通过真菌糖化酶（外切-1,4-α-葡糖苷酶，EC 3.2.1.3）将糖原水解为葡萄糖基单位，并采用葡萄糖氧化酶比色法测定葡萄糖。颜色强度可在传统酶标仪中测量，数据可输入在线计算机进行快速处理。该方法的优点在于其简单性和自动化程度，减少了样品处理步骤，与其他传统方法相比所需细胞数量较少。
