Assay Method Information |
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| Screening Assay for ABCG2 Inhibitors |
Description: | IC50 and maximal activities for inhibition of PhA accumulation were determined from dose-response curves. The accumulation of PhA, a fluorescent ABCG2 substrate, formed the basis of the assay for inhibitors of ABCG2 activity. NCI-H460/MX20 cells were transferred to black-walled, clear-bottomed 384-well polylysine-coated assay plates (Corning) and allowed to attach for several hours. PhA was added, immediately followed by compounds or vehicle (DMSO/PBS) control and incubated for an additional 18 h. After removal of medium and washing with PBS containing Ca2+ and Mg2+, fluorescence intensity was read on a Tecan Safire fluorescence plate reader in bottom read mode (395 nm excitation, 670 nm emission). Each plate had control wells containing Fumitremorgin C (FTC). Data were normalized to FTC and reported as the percentage of FTC fluorescence. Apparent IC50 values were calculated from dose-response data using SigmaPlot (SPSS, Inc., Chicago) 4-parameter logistic nonlinear regression analysis. |
Affinity data for this assay | |
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