Serum charcoal stripped

Fig. 4. The eflfect of charcoal-stripped plasma of various dilutions on the standard immunoassay curve for testosterone. Immunoassays were carried out as described in the text in plasma diluted with 0.1% bovine serum albumin.

The cells are grown in 96-well plates in estrogen-free medium (phenol red free, with charcoal-stripped calf serum) and contain test compounds and antiestrogens at concentrations that are varied over several log orders. For the antiestrogen assay, to the cells, a range of concentrations of samples are added concurrently with I-nM antiestrogens. After grown for 3 days, to determine AP activity, the cells are frozen, defrosted, and incubated with p-nitrophenylphosphate at room temperature, and the hydrolysis product p-nitrophenol is measured kinetically at 405 nm [50]. 

Breast cancer cell lines are known to proliferate in response to estrogen stimulation. Charcoal-dextran stripped human serum inhibits the proliferation of MCF-7 breast cancer cells in a dose-dependant fashion. Estrogens overcome this effect. The E-screen test exploits the estrogen-dependant proliferation of MCF-7 cells and has been used previously for the determination of the estrogenic potential of phenol red, estradiol, and tamoxifen among other compounds. Such assays can take 4-7 days. 

The N3 supplement is used as a serum substitute to avoid retinoids m serum triggering the response of reporter cells N3, however, can be arduous and expensive to prepare (see Table 1). Given the short-term nature of this assay, it may be advisable first to try the assay in serum-contaimng medium or in medium using serum that has been stripped of retinoids by treatment with charcoal followed by sterile filtration Alternatively, serum-free medium supplement containing insulin, transferrin, and selenite can be obtained from Boehnnger Mannheim (Indianapolis, IN) Whichever medium or serum treatment is chosen, it is important that the viability of both the tissue explant and reporter cell monolayer is ensured over the duration of the assay... 

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