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.
1. Human estrogen receptor β transfection assay
NOTE: Aseptic technique and a laminar flow hood is required for Day 1 of the assay protocol.
The authors have nothing to disclose.
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 |