Login
欢迎浏览恩派尔生物资料网
我要投稿 请登录 免费注册 安全退出

您现在的位置是: 首页 > 实验方法 > 细胞技术

细胞技术

Assessing Modulation of Estrogenic Activity of Environmental and Pharmaceutical Compounds Using MCF-

2025-03-06 细胞技术 加入收藏
The MCF-7 cell line was isolated from a pleural metastasis of a human breast ade

The MCF-7 cell line was isolated from a pleural metastasis of a human breast adenocarcinoma, and, when grown on plastic substrates, typically forms a continuous cell monolayer at confluence (1 ). MCF-7 cell cultures respond to 17β-estradiol (E2 ) by increases in the expression of a number of genes ((2 ),(3 ) and localized focal postconfluent cell proliferation, which results in development of multicellular, three dimensional nodules termed “foci” (4 ). Thus, focus development in MCF-7 cells may represent the basic characteristics of an estrogenic response, i.e., induction of concerted gene expression, resulting in tissue restructuring through enhanced postconfluent cell proliferation. Since foci are easily counted, the development E2 -induced foci and their inhibition are useful as a relevant human-tissue-based assay for the assessment of estrogenic and antiestrogenic activity of environmental and pharmaceutical compounds (5 -7 ). Here the authors give the protocol for measuring focus formation in response to estrogen-modulating agents. In addition, protocols are presented to determine whether the modulation of foci by a particular agent is a result of estrogen-receptor (ER)-dependent activity or changes in the level of E2 through alteration of E2 catabolism. Table 1 provides an overview of the three protocols. Table 1  Overview of Protocols for Assays a in cpm

 

Seed(cells/mL/well)

Refeed(after seeding)

Incubation with[3 H]E2

Endpoint

Measurement

MCF-7 Focus

1 � 105

24 h and every

None

Development of

Increase or

assay


3-4 d for a


foci

decrease in focal



total of 4


Inhibition of focus

retention of



refeeds


development

rhodamine B stain

Whole-cell

5 � 105

24 h

For 3-4 h; at the

Displacement of

Decrease of [3 H]E2

ER-binding



same time as test

[3 H]E2 from ER

in cells, with

assay



agent or a specified

by test agent

increase in test




time after test agent;


agent




at either 4 or 37�C



Radiometric

5 � 105

24 h

Between 3 and 24 h;

Increase in tritiated

Increase in tritiated

analysis of



after incubation with

catabolism of

H2 O in media,

catabolism of



test agent for a

[3 H]E2 , resulting

with increase

[3 H]E2



series of time points;

in production of

in test agent




at 37†C

Tritiated H2 O



文章底部广告位

文章评论

加载中~