In vitro protocols for estrogenic activity of chemicals and drugs
Recombinant yeast assay
I. For ER protein transformed into BJ3505 yeast, selection media is Minimal Media (MM) plus Lysin and Histidine
50 ml of 10X Yeast Nitrogen Base (YNB) 67g YNB/L
50 ml of 20% Dextrose (DEX) 20g dex/100ml
5 ml each of Lysine (LYS) 1.8g L-lysine-HCL/500ml
Histidine (HIS) 1.2g L-histidine-HCL/500ml
Add water to make 500 mls
Store at room temperature.
2. Selection plates for 100mls (making for from 4 to 5 plates of 100mm):
10 mls 10X YNB
78 mls water
Allow to be cool until the container can be handled without difficulty.
Add solution containing: 1 ml LYS (stock solution)
1 ml HIS (stock solution)
10 mls 20% DEX
Swirl to mix, Pour 15-20 mls per 100mm plate;
Let the plates stand at RT to harden and Store at 4°C
Receptor: ERtrp (YePtrpER) Reporter: E2.ura (YrpE2ura)
II. For AR/G2 protein transformed into YPH500, the selection media is the Minimal Media (MM.) plus Adenine., Lysine., Tryptophan., and Uracil (or Histidine)
1. Growth media:
50 mls of 10X Yeast Nitrogen Base (YNB) 67g YNB/L
50 mls of 20% dextrose (DEX) 20g DEX/100 ml
5 mls each of Lysine (LYS) 1.8g lysine-HCL/500 ml
Tryptophan (TRP) 2.4g TRP/500 ml
Histidine (HIS) 1.2g HIS/500 ml
17 mls of Adenine (ADE) 0.6 g adenine sulfate/500 ml
Enough water to make 500 mls
Filter to sterilize
Store at room temparature.
2. Selection plates – for 100 mls (make 4-5 plates of 100mm)
10 mls 10X YNB
2 g bactoagar
75.3 mls water;
Allow to be cool till the container can be handled without difficulty.
Add solution containing: 1 ml LYS 1 ml TRP
1 ml HIS (all from stock solutions)
10 mls 20%DEX
1.7 mls ADE from stock solution
Swirl to mix;
Pour 15-20 mls/100mm plate;
Let the plates stand at room temperature to harden.
Store at 4°C
Receptor: ARleu (Yep24leu2) Reporter: YRpG2.his
II. Stock solutions
1) 10X YNB, 67g/l, filter, store at room temparuate (RT)
2) 20% dextrose, 20g/100mls, filter, RT
3)1M CuSO4, 24.9g/100ml, filter, RT
4)Adenine sulfate, 0.6g/500ml, autoclave, RT
5)L-Histidine-HCl, 1.2g/500ml, autoclave, 4ºC
6)L-Lysine-HCl, 1.8g/500ml, autoclave, 4ºC
7) L-Tryptophan,2.4g/500ml, filter, 4ºC
Z buffer: 60mM Na2HPO4, 40mM NaH2PO4, 10 mM KCL, 1 mM MgSO4, PH 7.0
Zymolyase 20T 20,900U/g
dissolve to 25 U/g/ml= 1.163mg/ml=1163mg/l buffer
MCF-7 cell proliferation assay
1. MCF-7 cells (purchased, provided from cell line bank and stored in straws anchored in liquid nitrogen)
2. Phenol red-free D-media (EMEM with 50% increase of all essential amino acids except glutamine, 50% increase of all vitamins, and 100% increase of all non-essential amino acids) supplemented with 5% fetal bovine serum (FBS). Adjust pH, autoclave, filter and add PSN antibiotic mixture (3ml/l).
3. Cells are grown in phenol red-free D-media, incubated at 5% CO2, 95% air and 100% of humidity at was 37oC.
4. Chemicals or drugs are diluted in the phenol red- free D media with 5% dextran-coated charcoal-stripped FBS (DCC-FBS) and 3ml/l PSN (test media) (Ethanol 0.1% in test media should be used as the vehicle; estradiol benzoat or other well known estrogenic compound are used as positive controls).
5. The cells (5x104/ml) plated in 6-well culture plate (2ml/well), in triplicate, are allowed to attach for 24h. 6. The phenol red-free D media was replaced with phenol red-free D media supplemented with 5% DCC-FBS (incubation for 24h).
7. Remove the media and replace with the test media containing test extracts followed by incubation (37oC, 3 days, once change of the test media).
8. Harvest the cells by tripsin, dillute and count under microscope (with Newbauer’s chamber).
And/or measurement of DNA isolated from the cultured cells can be alternative .
9. Collect data for statistics.
Transient transfection assays
I. Plating and transfection
1. Plate HepG2 human hepatoma cells in quadruplicate in 24-well plates at a density of 5x104 cells/well in complete medium containing phenol red-free Eagle’s minimal essential medium supplemented with 10% DCC-FBS, 2% L-glutamine, and 0.1% sodium pyruvate (for 24 h at 37oC in a humidified atmosphere of 5% CO2 air).
2. Transfected step carried with two plasmids: (1) 0.4 mg/well receptor plasmid encoding rat ERa, (2) 0.8 mg/well C3-Luc, reporter plasmid.
3. Rinse the transfected cells with phosphate-buffered saline (PBS) and treat with various concentrations of chemicals or drugs (absolute alcohol and E2 are used as vehicle control and positive control, respectively) 4. After 24 h incubation, rinse the cells with PBS and lyse with 65 ml of passive lysis
4. Plate the lysate into 96-well plates for luciferase determination.
II. Dual Luciferase reporter assay
1. 100 ml of Luciferase assay reagent II (promega, Medison, WI, USA) was added into each well to 20 ml of lysate.
2. Determine immediately firely luciferase activity using microplate luminometer LB96P (Berthold technologies, Germany).
3. Add 100ml of Stop & Glo reagent (Promega, Medison, WI, USA) and measure Renilla luciferase activity using the DLRTM Assay System (Promega, Medison, WI, USA).
4. The luminescence from the firefly luciferase reaction is ‘experimental’ reporter and the luminescent reaction of Renilla luciferase is ‘control’ reporter.
5. Collect data for statistics!