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. Prepare dilutions of 17β-Estradiol for the standard curve. Transfer the Cell Recovery Medium and Compound Screening Medium (CSM) from the freezer storage and thaw them in a 37 °C water bath. Label microcentrifuge tubes Intermediate 1 and 2 (INT1, INT2) and 1-8. Fill …
Disclosures
The authors have nothing to disclose.
Materials
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