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Assay Method Information

Assay Name:  ER Transcription Assay (MCF7 Cells)
Description:  The ER transcription assay is a reporter assay that is based on the ability of ER to induce transcription from a luciferase reporter gene containing estrogen response elements (EREs) in the promoter/enhancer region. When the reporter gene is transfected in MCF7 cells (containing endogenous ER), transcription is reflected by the level of luciferase expression.MCF7 cells are maintained in DMEM/F12 (Gibco, catalog number 11330) supplemented with 10% fetal bovine serum (FBS) (Gemini Bio-Products, catalog number 100-106). A day before transfection, cells are split into a T75 flask at a cell density of 300,000 cells/mL (10 mL total) and allowed to attach overnight in a humidified CO2 incubator at 37° C.Next day, prior to transfection, media is switched to DMEM/F12 (Gibco, catalog number 21041) supplemented with 10% charcoal-stripped serum (Gemini Bio-Products, catalog number 100-119). MCF7 cells are then bulk transfected, using Lipofectin (Invitrogen, catalog number 18292) with the following plasmids: 7x-TK-ERE-Luc3 (ER reporter gene) and pCMV-Renilla (normalization control). Briefly, for each T75 flask, 32.5 μL of Lipofectin is added to 617.5 μL of OptiMEM (Gibco #11058) and incubated for 30 min at 37 C. Approximately 20 ug DNA is mixed in OptiMEM (Invitrogen) to a total volume of 650 μL. Following incubation, the OptiMEM-DNA mixture is added to the OptiMEM-Lipofectin mix and incubated for 15 minutes at 37° C. The DNA-Lipofectin mixture is then added directly to the T75 flask and the flask is returned to the incubator.After overnight incubation, compound is added to individual wells of a 96-well plate in a 10 μL volume of media at 10× concentration along with 1713 estradiol whose final concentration is 0.1 nM. Normally, DMSO (used as a vehicle) is included to achieve a final concentration of 0.1% when added to the cells. Transfected cells are trypsinized, resuspended in DMEM/F12/10% charcoal-stripped serum and added to the 96-well plate at 25,000 cells/well in 90 μL of media. The plate is then returned to the incubator for 24 hours.After incubation with compounds for 24 hours, Firefly and Renilla luciferase activities are measured to determine ER transcriptional activity. Media is removed from 96-well plates by decanting and blotting on paper towels. Cells are lysed with 40 ul/well of 1× passive lysis buffer (25 mM Tris Phosphate, 2 mM CDTA, 10% Glycerol, 0.5% Triton X-100 and 2 mM DTT before use) and allowed incubate at room temperature for 10 minutes.Firefly luciferase activity is measured by adding 30 ul Firefly luciferase assay buffer (20 mM Tricine, 0.1 mM EDTA, 1.07 mM (MgCO3)4 Mg(OH)2*5H2O, 2.67 mM MgSO4, 33.3 mM DTT, 270 μM Coenzyme A, 470 μM luciferin, 530 μM ATP, reconstituted) per well, followed by measuring light units using a luminometer (BMG labtech FLUOstar OPTIMA). One second total read time after a one second delay.Renilla luciferase activity is measured by adding 50 ul Renilla luciferase assay buffer (1.1M NaCl, 2.2 mM Na2EDTA, 0.22 M KxPO4 (pH 5.1), 0.44 mg/mL BSA, 1.3 mM NaN3, 1.43 uM coelenterazine, final pH adjusted to 5.0), per well, followed by measuring light units using a luminometer. One second total read time after one second delay. If Firefly luciferase signal is high, Renilla assay must be done an hour after the Firefly assay due to incomplete squelching of Firefly signal.
Affinity data for this assay
 

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Last update November 1, 2007
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