Estrogen Receptor-Reporter Activity Assay to Screen Phytoestrogen Estrogenic Activity

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.
   4. Transfer 5 μL of 100 μM 17β-Estradiol Stock into INT1. Discard the tip. Vortex.
   5. Before each transfer, rinse the pipette 3 times, and then transfer 10 μL from INT1 into INT2. Discard the tip.
   6. Rinse the pipette 3 times, and then transfer 100 μL from INT2 into tube 1. Discard tip. Transfer 300 μL from tube 1 into tube 2. Repeat for tubes 3 through 7. Discard 300 μL from tube 7 into the waste container. Tube 8 is a Zero and does not receive estradiol. Final concentrations of plated standards are: 400, 133.3, 44.44, 14.815, 4.938, 1.646, 0.5487, and 0 pM estradiol.
2. Prepare sample compounds.
   1. Vortex samples.
   2. Take 4 μL of each plant sample in DMSO and add to 496 μL of CSM to yield a 0.8% DMSO solution.
3. Rapidly thaw Reporter Cells.
   1. Retrieve the tube of Cell Recovery Medium from the 37 °C water bath. Disinfect the outside surface using 70% ethanol.
   2. Retrieve Reporter Cells from -80 °C storage and thaw by transferring 10 mL of the pre-warmed CRM into the tube of frozen cells.
   3. Close the tube of Reporter Cells and transfer them to a 37°C water bath for 5-10 min.
   4. Retrieve the tube of Reporter Cell Suspension from the water bath. Invert the tube of cells several times gently to break up aggregates of cells and produce a homogenous suspension. Clean the surface of the tube with 70% ethanol.
4. Assay plating.
   1. Dispense 100 μL of the Reporter Cell Suspension into each well using a multichannel pipette.
   2. Dispense 100 μL of samples in triplicate into appropriate assay wells.
   3. Transfer the plate into a 37 °C, humidified 5% CO₂ incubator for 22-24 h.
5. Thaw Detection Substrate and Detection Buffer in a dark refrigerator overnight to prepare for Day 2.
6. Just prior to the end of the plate incubation, remove Detection Substrate and Detection Buffer from the refrigerator and place in a low light area until equilibrated to RT. Once at RT, invert each tube gently several times to thoroughly mix solutions.
   1. Immediately before the incubation is complete, pour the entire contents of the Detection Buffer into the tube of Detection Substrate to create Luciferase Detection Reagent. Mix gently so as not to produce foam.
   2. Once the incubation is complete, invert the plate to discard contents into an appropriate waste container. Gently tap the plate on a clean absorbent paper towel to remove the last droplets from the wells.
   3. Add 100 μL of the Luciferase Detection Reagent to each well. Allow the assay plate to rest at RT for 15 min. Do not shake the plate.
7. Quantify luminescence using a 96-well plate-reading luminometer.