Estrogen Receptor-Reporter Activity Assay to Screen Phytoestrogen Estrogenic Activity

Abstract

Source: Fender, E. et al., Screening for Phytoestrogens using a Cell-based Estrogen Receptor β Reporter Assay. J. Vis. Exp. (2021).

This video describes a cell-based reporter assay to determine the estrogenic activity of plant-derived compounds. A compound with estrogen-like activity mimics its binding to estrogen receptors and drives downstream reporter enzyme gene transcription. The reporter enzyme generates bioluminescence in a reaction, indicating the estrogenic activity of the compound.

Protocol

1. Human estrogen receptor β transfection assay

NOTE: Aseptic technique and a laminar flow hood is required for Day 1 of the assay protocol.

1. Prepare dilutions of 17β-Estradiol for the standard curve.
   1. Transfer the Cell Recovery Medium and Compound Screening Medium (CSM)   from the freezer storage and thaw them in a 37 °C water bath.
   2. Label microcentrifuge tubes Intermediate 1 and 2 (INT1, INT2) and 1-8.
   3. Fill INT1 with 995 μL of CSM, INT2 with 615 μL of CSM, tube 1 with 900 μL of CSM, and tubes 2-8 with 600 μL of CSM. Set tube 8 aside.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1000 µL pipette
20 µL pipette
200 µL pipette
37 °C water bath
37 °C, humidified 5% CO2 incubator
70% ethanol
Analytical balance
Cell culture-rated laminar flow hood
Dimethyl sulfoxide
Disposable media basin, sterile
Drip filtration system
Erlenmeyer flasks			125 mL and 250 mL
HPLC grade methanol
Human ERβ Reporter Assay System, 1 x 96-well format assays	Indigo Biosciences	IB00411	Assay kit - analyzes 24 samples plus standard curve
Lyophilizer
Multi-channel pipette
Orbital shaker
Plate-reading luminometer			ex. Bioteck Synergy HTX
Rotary evaporator
Round bottom flasks			50 mL and 300 mL
Sterile microcentrifuge tubes or sterile multi-channel media basins
Sterile tips			200 µL and 1000 µL
Whatman grade 1 paper
Whirl-pak bags			sterile polyethylene bags