DETERMINATION OF THE NUMBER OF ENDOTHELIAL-CELLS IN CULTURE USING AN ACID-PHOSPHATASE ASSAY
DETERMINATION OF THE NUMBER OF ENDOTHELIAL-CELLS IN CULTURE USING AN ACID-PHOSPHATASE ASSAY
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DOI：
10.1016/0003-2697(86)90131-4
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发表时间：
1986-01-01
影响因子：
2.9
通讯作者：
FEDER, J
中科院分区：
文献类型：
--
作者：
CONNOLLY, DT;KNIGHT, MB;FEDER, J
描述了一种简单的测定方法，其中少量的培养中的内皮细胞可以通过测量酸性磷酸酶活性来确定。从在96孔培养板中生长的细胞中除去生长培养基后，将细胞在含有去污剂Triton X-100和磷酸酶底物磷酸对硝基苯基酯的缓冲液中裂解。在37 ℃下2小时后，C，用氢氧化钠终止反应，并使用快速多孔板读数器测定显色。该测定法每孔检测100至10，000个细胞。该测定已被用来确定在存在和不存在的内皮细胞生长因子从牛下丘脑的内皮细胞的生长曲线，并在生长因子的纯化过程中监测馏分。对测定进行微小修改，使其完全自动化。
A simple assay is described in which small numbers of endothelial cells in culture can be determined by measuring acid phosphatase activity. After removal of the growth medium from cells grown in 96-well culture plates, the cells are lysed in buffer containing the detergent Triton X-100 and the phosphatase substrate p-nitrophenyl phosphate. After 2 h after 37.degree. C, the reaction is stopped with sodium hydroxide, and color development is determined using a rapid multiwell plate reader. The assay detects 100 to 10,000 cells per well. The assay has been used to determine growth curves for endothelial cells in the presence and absence of endothelial cell growth factor from bovine hypothalamus and to monitor fractions during purification of the growth factor. Minor modifications in the assay allow it to be fully automated.