Consumer and Health Products Safety Thermo Scientific ToxInsight

Thermo Fisher Scientific Inc. Pittsburgh, Pennsylvania (412) 770-2200

100 Technology Drive

15219 USA www.thermoscientific.com/cellomics

R04-1001-ERa ToxInsight Estrogen Receptor α Cartridge, materials for 7 96-wells (6 compounds)

R04-1001-AR ToxInsight Androgen Receptor Cartridge, materials for 7 96-wells (6 compounds)

Kit Contents: R04-1001-ERa R04-1001-AR

ERα CryoRedi cells 7 (box 2) --

AR CryoRedi cells -- 7 (box 2)

Hoechst 33342 30 L 30 L

10x Perm Buffer 100 mL 100 mL

10x Wash Buffer 100 mL 100 mL

Nunc EDGE Plates 7 7

Media 500 mL 500mL

Media supplements:

Serum

Supplement 1

Supplement 2

(box 2) (1) 25 mL (2) 5 mL

(2) 2.5 mL

(box 2) (1) 25 mL (2) 5 mL

(2) 2.5 mL

Bottles for Assay/Plating Media 2 2

Thin Plate Seal Assembly 1 x 7/pack 1 x 7/pack

STORAGE: Kit will arrive in two separate packages. For Package #1, store all buffers, media, and dyes at 4°C protected

from light. For Package #2, store the cells in vapor phase of liquid nitrogen and media supplements at -20°C. Allow the media, supplements, serum, buffer, and dyes to warm to room temperature before use. See the Solution Preparation section

for storage and stability of prepared solutions.

WARNING: Please completely read these instructions and the accompanying material safety data sheets before using this

product. Thermo Scientific Reagents are not for diagnostic use in humans or animals.

PLEASE NOTE: If you are testing where regulation requires you to document proper routine instrumentation calibration,

please refer to the ToxInsight Illumination Correction and System Calibration Guide and documentation sheets for additional

information. We suggest that you run these tests PRIOR to setting up the biological protocols. If you would like to perform

routine system calibrations, we suggest that you order the following:

K10-0099-1 Thermo Scientific OptiTracker Optical Performance Monitoring Kit

Endocrine disruptors are compounds that alter steroid activity thereby perturbing endocrine system functionality.

Environmental endocrine disruptors have been linked to numerous adverse health effects and reproductive problems in both

humans and wildlife. The advent of complex chemical libraries in addition to a large catalog of existing compounds

necessitates an automated procedure to assess a chemical‟s in vitro endocrine activity prior to investigating potential

organismal and environmental impact. Utilization of in vitro screens for potential endocrine active chemicals (EACs) can

reduce animal testing by categorizing and prioritizing chemicals based on their ability to alter endocrine receptor activity.

Bioethics, efficiency, and cost of animal testing have been moving the trend towards in vitro cell-based assays to replace

protocols using animal tissues, to reduce animal usage by predicting potential in vivo action, and to provide additional

toxicity information. The need for more sensitive assays that can provide insight into mechanisms of action for endocrine

receptor activation extends to environmental and industrial safety as well as consumer products from cosmetics to food

additives. A goal of in vitro testing is to detect compounds with potential endocrine activity and assess the magnitude and

Consumer and Health Products Safety – Thermo Scientific

ToxInsight Endocrine Profiler Panel Cartridge:

Estrogen Receptor α (alpha) and Androgen Receptor Assays

High-Content Imaging Reagents




potency of the response. Because of diverse action mechanisms with different compounds, monitoring multiple, independent

outputs in the same cell increases the predictive power of the assay.

Thermo Scientific Redistribution Technology

Thermo Scientific Redistribution technology monitors the cellular translocation of GFP-tagged proteins in response to

compounds or other stimuli and allows easy acquisition of multiple readouts from the same cell in a single assay run. In

addition to the primary readout, high content assays provide supplementary information about cell morphology, fluorescence

localization, and cellular toxicity. Thermo Scientific CryoRedi cell-based assays utilize Redistribution technology from

frozen cells which can be thawed, plated, and used directly in high content analysis applications, including profiling and

screening studies.

The ERα Redistribution Assay Estrogen is a regulator of normal endocrine functions. Signal transduction induced by estrogens such as estradiol, the main

endogenous human estrogen, is mediated by the estrogen receptor (ER). ER is a nuclear receptor that upon ligand binding

organizes into homo- and heterodimers of the ERα and ERβ subtypes. Following ligand binding, ER acts as a transcription

factor and regulates expression of several target genes such as cyclin D1 and IGF-1 [1]. Estrogens and estrogen receptors are

implicated in development and progression of breast cancer. Moreover, environmental chemical contaminants with estrogenic

activity are suggested to promote reproductive disorders. The ERα Redistribution Assay monitors accumulation of an EGFP-

ERα fusion protein in nuclear foci as a measure of ERα activation (Figure 1). The agonist version of the assay uses the potent

estrogen 17β-estradiol as a reference compound.

Figure 1. Illustration of ERα Redistribution upon stimulation with 17β-estradiol

The AR Redistribution Assay The androgen receptor (AR) is a nuclear receptor transcription factor that mediates the cellular actions of androgens, the male

sex steroids testosterone and dihydrotestosterone (DHT). Androgen signaling mediates male sexual differentiation, sexual

maturation, and spermatogenesis. Androgens maintain normal prostate homeostasis but are also involved in prostate

tumorigenesis [2]. Androgens exert their activity through binding to AR resulting in either activation or repression of

androgen responsive genes. Ligand binding exposes a nuclear localization signal (NLS) allowing translocation of AR to the

nucleus via a transport protein. Agonists as well as most antagonists induce translocation of AR to the nucleus. However,

only agonists activate AR functions by interaction with androgen response elements (AREs). Moreover, AR agonists often

induce nuclear localization of AR with a punctuate distribution pattern, the so-called nuclear foci [2, 3, 4]. The AR

Redistribution Assay is designed to assay for compounds inducing EGFP-AR nuclear translocation and formation of nuclear

foci. AR nuclear accumulation and foci formation is promoted by the agonist DHT, which is used as reference compound

(Figure 2).

Figure 2. Illustration of AR Redistribution upon stimulation with DHT

The ToxInsight Endocrine Profiler Panel (EPP) Assay Cartridge identifies compounds that have the potential to cause endocrine

disruption by simultaneously monitoring of multiplexed targets including cell loss, nuclear DNA, morphological

characteristics, and nuclear receptor foci formation, along with potential degradation (for ERα) or translocation of receptor

(AR). These cartridges use an end-point assay based on Redistribution technology and fluorescence detection in fixed cells

grown on standard high-density microplates. The DNA binding dye, Hoechst 33342, is used to identify nuclei and monitor

viability (cell number), morphology, and nuclear intensity outputs. Under normal (untreated) conditions, GFP appears to be

Unstimulated cell:

EGFP-ERα localized in the nucleus

Stimulated cell:

EGFP-ERα localized in the nuclear foci

17β-estradiol

Stimulated cell:

EGFP-AR localized in the nucleus. Foci formation evident

DHT

Unstimulated cell: EGFP-AR localized both

in the cytoplasm & nucleus




evenly distributed (within the nuclear region for ERα and throughout the cytoplasm and nuclear region for AR). For ERα

cells, upon treatment with β-estradiol, foci are formed within the nuclear region, followed by degradation within the nuclear

region (seen as an overall decrease in total nuclear GFP intensity; Figure 3, left). In AR cells, treatment with DHT also has

foci formation, but also exhibits translocation from cytoplasm to the nuclear region (Figure 3, right).

Figure 3. Inset visualization of GFP foci in ERα (A) and AR (B) cells (20X objective). Cells treated with β-estradiol (A, bottom) or DHT (B, bottom) show an accumulation of spots within the nuclear region. AR cells also show translocation from the cytoplasm to the nucleus. Images were acquired using the Thermo Scientific ToxInsight IVT Reader. Overlays in the bottom right images represent visualization of the Compartmental Analysis BioApplication to identify spots (green, A & B) and cytoplasmic area for calculating translocation (orange, B).

A goal of in vitro testing for potential endocrine disruptors is to ultimately generate data that can be correlated to in vivo

effects. Because of diverse action mechanisms with different compounds, monitoring multiple, independent indicators of both toxicity and receptor responses in the same cell increases the predictive power of the assay. The protocols in this

instruction booklet pertain to measuring both the Dose Range for toxicity and Receptor Activity using either ERα or AR

cells. The data acquired using the EPP Assay Cartridge should be analyzed with the provided Thermo Scientific AddIn for

Microsoft® Excel® and Excel Template for the EPP Assay to predict endocrine disruption from the tested compounds.

Dimethyl Sulfoxide (DMSO; suggested vendor – Sigma #D2438, CAS 76-68-5)

16% Paraformaldehyde (suggested vendor – EMS #15710, CAS 50-00-0)

Paraformaldehyde will be diluted to final concentration of 4%

We do not recommend that you prepare fix solution by diluting from a 37% formaldehyde solution

Positive and weak positive compounds

For ERα assays, we suggest using β-estradiol (Sigma, #E8875, CAS 50-28-2) and Bisphenol A (BPA, Sigma,

#239658, CAS 80-05-7) for positive and weak response ERα controls, respectively

For AR assays, we suggest using DHT (Sigma, # A8380, CAS 521-18-6) and progesterone (Sigma, #P0130,

CAS 57-83-0) for positive and weak response AR controls, respectively

You will need a Schedule 3 license from the DEA in order to use DHT (or other Schedule 3

compounds within the United States). Please see http://www.deadiversion.usdoj.gov/drugreg/reg_apps/onlineforms_new.htm for more information (note that each state has different requirements). For customers outside the

United States, please consult proper regulatory guidelines for handling Schedule 3 compounds.

Sterile distilled H2O for moat in plates (suggested vendor – Thermo Scientific HyClone cell culture products

# SH30529.02)

Distilled deionized H2O for buffer dilutions

Liquid Nitrogen Dewar (or equivalent) for storing cells prior to use

A: ERα cells B: AR cells

GFP GFP CircSpot Ring; CircSpot

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Two types of media are used for this assay: Plating Media and Assay Media. Media should be kept at 4°C until use.

Supplements and serum are to be kept at -20°C until use. When preparing each media, ensure that the supplements and serum are completely thawed before use.

For Plating Media:

1. Aliquot 220 mL of provided media into one of the 250 mL bottles.

2. Add Serum.

3. Add (1) vial of Supplement 1.


5. Mix gently. Add label and fill in information.

For Assay Media:

1. Aliquot 245 mL of provided media into the other 250 mL bottles.



4. Mix gently. Add label and fill in information.

When complete, store media at 4°C until use.

Label information – We suggest for each label that you add the name of the person who prepared the media, the date of

adding the supplements, and suggested expiration date. Expiration date should be either earliest expiration date on

provided material or 1 month after opening, whichever is first.

1. Warm Plating Media to room temperature prior to use (do not heat to 37oC).

2. CryoRedi cells allows you to plate cells directly from liquid nitrogen. No passage is required. Cells should be in the

vapor phase of liquid nitrogen until use.

3. When setting up the assay, remove vials from liquid nitrogen and thaw cells for 1-3 minutes in a 37oC waterbath.

4. Transfer cells from vial to 15 mL conical tube.

a. Note: For Activation Assays, if using more than 1 vial at one time, they can be combined into a 50 mL

conical or multiple 15mL conicals. Adjust volume on a „per vial‟ basis.

5. Add 5 mL of Plating Media per vial slowly into conical.

6. Centrifuge for 3 minutes at 250 x g.

7. Remove supernatant and re-suspend the cells in 5 mL of Plating Media per vial. 8. Count cells using hemacytometer.

9. Further dilute cells in Plating Media so that 8000 cells per 100 µL per well in Nunc Edge plates.

a. We suggest that you have 11-12 mL total of media + cells in order to add 100 µL per well per plate. For

example, if hemacytometer count = 200,000 cells/mL and you have 5 mL already, you have a total of

1,000,000 cells (200,000 x 5). If you would want to plate cells in 11 mL of media (=880,000 cells needed),

you would add 4.4 mL of your cell media to 6.6 mL of Plating Media (if you would like 12 mL for plating,

you would use 4.8 mL of cell media to 7.2 mL Plating Media).

10. Fill Nunc Edge moats with 2 mL of sterile water per quadrant.

11. Let plate rest at room temperature on vibration-free table for 1 hour.

12. Incubate plate overnight at 37oC.




Keep media, buffers, and dyes at room temperature before use.

Be sure to warm fix solution before use.

We recommend that you make Stock Solutions fresh each time. If you choose to freeze stock solutions for future use,

please follow vendor recommendations.

We recommend that you implement automation (e.g., Matrix multichannel pipettor or Multidrop) to decrease variability.

See Figure 4 for general assay workflow; Tables 1-5 for reagent and compound preparation.

The approximate absorption/emission maxima of the fluorescent dyes are as follows:

Hoechst dye = 386/440 nm

GFP = 485/521 nm

Figure 4. General Workflow for EPP Assay

Table 1. Recommended Control Compounds for Running the EPP Assay. Positive and Weak Response

Controls should be adhered for proper Dose Range and Activity Assays (as setup in Figures 5 and 6).

Compound Vendor Cat# CAS# Type

β-estradiol Sigma E8875 50-28-2 ERα Positive Control

Bisphenol A Sigma 239658 80-05-7 ERα Weak Response Control

DHT Sigma A8380 521-18-6 AR Positive Control

Progesterone Sigma P0130 57-83-0 AR Weak Response Control




Table 2. Solution Preparation – Dose Range Assays (per 96-well plate unless noted). See Tables 1 and 3 for additional

information. It is suggested to make all reagents at the time of experimentation.

Assay Media for dilutions (=DMSO Media) (Per Plate)

We suggest that you make Assay Media for compound dilutions already in 2% DMSO for easy dilution.

24.5 mL Assay Media + 500 µL DMSO should be sufficient per plate.

Vehicle Control Solution (Per Plate)

Add DMSO to Assay Media for a final volume of 2%. For Dose Range assay, we suggest that you make up 980 µL Assay Media + 20 µL DMSO.

Positive Control Stock Solution

(For both Dose Range and Activity Assays)

For β-estradiol: Suggestion for Sigma #E8875 is to make up as 10 mM stock in DMSO (i.e., if you weigh out 5 mg of chemical, dissolve in 1.84 ml of DMSO [5 mg ÷ 272.4 mg/mmol ÷ 10-2 mmol/ml]).

For DHT: Suggestion for Sigma #A8380 is to make up as 10 mM stock in DMSO (i.e., if you weigh out 5 mg of chemical, dissolve in 1.72 ml of DMSO [5 mg ÷ 290.4 mg/mmol ÷ 10-2 mmol/ml]).

Positive Control Working Solution

(Per Plate)

Working solution should be made at 2X so that 100 µL is added to each well.

For β-estradiol: 50 nM final (make up as 100 nM); #E8875 is made up as 10 mM stock, so for

constant DMSO concentrations, a working stock of 5 µM is suggested. Two possible dilution schemes: 1. Dilute 1 µL 10 mM stock + 9 µL DMSO (= 1 mM) and then 2 µL of dilution + 398 µL

DMSO (= 5 µM) - preferred OR

2. Dilute 1 µL 10 mM stock + 1999 µL DMSO (= 5 µM) Final concentration will have 20 µL of 5 µM + 980 µL Assay Media.

For DHT: 50 nM final (make up as 100 nM); #A8380 is made up as 10 mM stock, so for constant DMSO concentrations, a working stock of 5 µM is suggested. Use same dilution as for β-estradiol.

Sample Stock Solutions (For both Dose Range and Activity Assays)

Follow suggested concentrations, keeping final diluted sample concentration at or below 1% DMSO[5]. Test solubility by making at a 10 mM stock solution; if found to be insoluble, dilute in half until soluble (i.e., test solubility at 5 mM).

Sample Working Solution (For Dose Range Assay)

(Per Plate)

Maximum final concentration for all compounds is 100 µM (make as 200 µM).

Add 10 µL 10mM stock + 490 µL Assay Media for 200 µM Max concentration. Perform 1:10 dilutions (50 µL of Max + 450 µL DMSO Media; 50 µL dilution + 450 µL DMSO Media, etc.).

1x Wash Buffer

(Per Plate)

Add 4 mL 10x Wash Buffer to 36 mL distilled water.

1x Staining Solution (Per Plate)

Add 1.5 mL 10x Perm Buffer to 13.5 mL distilled water to make 1x.

Add 1.5 µL Hoechst to 15 mL 1x Perm Buffer.

Fix Solution

(Per Plate) Dilute to 4% by adding 3.5 mL of 16% Paraformaldehyde in 10.5 mL 1x Wash Buffer.

Note: We do not recommend that you prepare fix solution by diluting from 37% formaldehyde.

Table 3. Example dilutions for Dose Range Assay

Compound Working

Conc

Final

Conc

First Concentration Dilutions

β-estradiol 100 nM 50 nM Using 10 mM stock, dilute to 5 µM (1:2000)

Either: 1 µL 10mM stock + 1999 µL DMSO (5 µM)

Or: Dilute 1 µL 10mM stock + 9 µL DMSO (1 mM) and then 2 µL of dilution + 398 µL DMSO (5 µM)

Then dilute to final concentration (1:50) = 20 µL (5 µM) dilution + 980 µL Assay Media

NONE

DHT 100 nM 50 nM Using 10 mM stock, dilute to 5 µM (1:2000)

Either: 1 µL 10mM stock + 1999 µL DMSO (5 µM)

Or: Dilute 1 µL 10mM stock + 9 µL DMSO (1 mM) and then 2 µL of dilution + 398 µL DMSO (5 µM)

Then dilute to final concentration (1:50) = 20 µL (5 µM) dilution + 980 µL Assay Media

NONE

Vehicle 2% 1% Add 20 µL DMSO + 980 µL Assay Media NONE

Sample 200 µM 100 µM 10 µL (10mM) stock + 490 µL Assay Media For 1:10 dilution = 50 µL dilution + 450 µL DMSO Media; repeat for each

dilution (see Figure 5)




Table 4. Solution Preparation - Activity Assays (per two 96-well plates as assay is performed in duplicate). See Tables 1

and 5 for additional information. We suggest that you make all reagents at the time of experimentation.

Assay Media for dilutions (=DMSO Media)

(Per 2 Plates)

We suggest that you make Assay Media for compound dilutions already in 2% DMSO for easy dilution.

Per 2 Plates, 29.4 mL Assay Media + 600 µL DMSO should be sufficient.

Vehicle Control Solution

(Per 2 Plates)

Add DMSO to Assay Media for a final volume of 2%. For Activity assay, make up 1.96 mL + 40 µL per 2 plates.

Positive Control Working Solution

(Per 2 Plates)


For β-estradiol: 50 nM final (make up as 100nM). First dilute from 10 mM stock to 5 µM as

noted above in the Dose Range preparation section. Per 2 plates, there will be 16 wells at the

highest dose (for Max Positive wells) + 4 wells needed for the starting dose for the Positive

dose response).

Max Positive concentration will have 50 µL of 5 µM working dilution + 2450 µL Assay Media

(100 µL per well will go into column 11 and into wells G2-H2). See Figure 5 for setup.

For Positive dose response dilutions, remaining wells will be a 1:5 dilution (200 µL of Max

Positive concentration + 800 µL DMSO Media; then 200 µL of dilution + 800 µL DMSO

Media, etc.).

For DHT: 50 nM final (make up as 100 nM)

Use same dilution suggestions as β-estradiol.

Weak Response Control Stock Solution

For BPA: Suggestion for Sigma #239658 is to make up 10 mM in DMSO

(i.e., if you weigh out 5 mg of chemical, dissolve in 2.19 ml of DMSO [5 mg ÷ 228.3 mg/mmol ÷ 10-2 mmol/ml]).

For Progesterone: Suggestion for Sigma #P0130 is to make up 10 mM in DMSO

(i.e., if you weigh out 5 mg of chemical, dissolve in 1.59 ml of DMSO [5 mg ÷ 314.5 mg/mmol ÷ 10-2 mmol/ml]).

Weak Response Control Working Solution

(Per 2 Plates)


For BPA: 500 nM final (make up as 1 µM). First dilute from 10 mM stock to 50 µM (1:200) as 1 µL stock +199 µL DMSO and then dilute to final concentration (1:50).

Per 2 plates, there will be 16 wells at the same concentration = 40 µL dilution + 1960 µL Assay

Media.

For Progesterone: 125 nM final (make up as 250 nM). First dilute from 10 mM stock to

12.5 µM (1:800) as 1 µL stock + 799 µL DMSO and then dilute to final concentration (1:50).

Per 2 plates, there will be 16 wells at the same concentration = 40 µL dilution + 1960 µL Assay

Media.

Sample Working Solution

(Per 2 Plates)

Maximum final concentration for the Activity Assay will be determined from running the Dose

Range Plate (see Section D for example). Concentration should be calculated at 2X the volume (as it is being added on top of the plating media in the wells). Remember that DMSO

concentration should remain the same. Please see Table 5 for example dilutions to ensure

proper DMSO concentrations are attained.

Concentration should be calculated at 1 mL total volume per compound for Max concentration

and 800 µL DMSO Media + 200 µL dilutions (1:5) as you will be doing triplicate wells in

duplicate plates. See Table 5 below for examples.

1x Wash Buffer

(Per 2 Plates)

Add 7.5 mL 10x Wash Buffer to 67.5 mL distilled water.

1x Staining Solution (Per 2 Plates)

Add 3 mL 10x Perm Buffer to 27 mL distilled water to make 1x.

Add 3 µL Hoechst to 30 mL 1x Perm Buffer.

Fix Solution (Per 2 Plates)

Dilute to 4% by adding 7 mL of 16% Paraformaldehyde in 21 mL 1x Wash Buffer.

Note: We do not recommend that you prepare fix solution by diluting from 37% formaldehyde.




Table 5. Example dilutions for Activity Assay. Note that concentrations are per 2 plates; therefore, for positive and weak

response compounds, you may need to adjust the volumes if evaluating more than 2 compounds.

Compound Working

Conc

Final

Conc

First Concentration Dilutions

β-estradiol 100 nM 50 nM Dilute 10 mM stock to 5 µM (1:2000)

Either: 1 µL 10 mM stock + 1999 µL DMSO

(5 µM)

Or: Dilute 1 µL 10 mM stock + 9 µL DMSO (1 mM) and then 2 µL of dilution + 398 µL DMSO (5 µM)

Then dilute to final concentration (1:50)

Per 2 plates (18 wells at highest dose with dilution) = 50 µL (5 µM) dilution + 2450 µL Assay Media

Per 2 plates for 1:5 dilutions, need 200 µL of highest dose + 800 µL DMSO Media; repeat for each dilution (see Figure 6)

BPA 1 µM 500 nM Dilute 10 mM stock to 50 µM (1:200) 1 µL 10 mM stock + 199 µL DMSO

Then dilute to final concentration (1:50) Per 2 plates (16 wells) = 40 µL (50 µM) dilution + 1960 µL media)

NONE

DHT 100 nM 50 nM Dilute 10 mM stock to 5 µM (1:2000)

Either: 1 µL 10 mM stock + 1999 µL DMSO

(5 µM)

Or: Dilute 1 µL 10 mM stock + 9 µL DMSO (1 mM) and then 2 µL of dilution + 398 µL DMSO (5 µM)

Then dilute to final concentration (1:50) Per 2 plates (18 wells at highest dose with dilution) = 50 µL (5 µM) dilution + 2450 µL Assay Media

Per 2 plates for 1:5 dilutions, need 200 µL of highest dose + 800 µL

DMSO Media; repeat for each dilution (see Figure 6)

Progesterone 250 nM 125 nM Dilute 10 mM stock to 12.5 µM (1:800)

1 µL 10 mM stock + 799 µL DMSO

Then dilute to final concentration (1:50)

Per 2 plates (16 wells) = 40 µL (12.5 µM) dilution + 1960 µL Assay Media

NONE

Sample *

200 µM 100 µM Per 2 plates = dilute directly to final concentration: 20 µL (10mM) stock + 980 µL media

Per 2 plates for 1:5 dilutions = 200 µL of dilution + 800 µL DMSO Media; repeat for each dilution (see Figure 6)

20 µM 10 µM Dilute stock to 1 mM (i.e., 3 µL of 10 mM stock +

27 µL DMSO)

Then dilute to final concentration (1:50) Per 2 plates = 20 µL (1mM) dilution + 980 µL Assay

Media

Per 2 plates for 1:5 dilutions = 200 µL

of dilution + 800 µL DMSO Media; repeat for each dilution (see Figure 6)

2 µM 1 µM Dilute stock to 100 µM (i.e., 1 µL of 10 mM stock + 99 µL DMSO) Then dilute to final concentration (1:50) Per 2 plates = 20 µL (100µM) dilution + 980 µL Assay Media


0.2 µM 0.1 µM Dilute stock to 10 µM (i.e., 1 µL of 10 mM stock + 999 µL DMSO)

Then dilute to final concentration (1:50) Per 2 plates = 20 µL (10 µM) dilution + 980 µL Assay Media


0.02 µM 0.01 µM Dilute stock to 1 µM (i.e., 1 µL of 10 mM stock + 99 µL DMSO; then take 1 µL of dilution + 99 µL DMSO)

Then dilute to final concentration (1:50) Per 2 plates = 20 µL (1 µM) dilution + 980 µL Assay Media


“*” indicates possible starting points as determined from Dose Range Assay. If stock concentration is not at 10 mM, you would

need to adjust accordingly.




Figures 5 and 6 are representative plate setups for both assay designs. The Dose Range assay plate must be setup,

completed, and run first before setting up the Activity plates. Actual Assay Procedures are similar for both types except for the starting concentration of each. The Dose Range plate should start with test chemicals at 100 µM, while the

Activity plate starting concentration per test chemical is dependent upon the values attained from the Dose Range plate

(see Figure 8 for example).

Figure 5. Suggested plate setup for Dose Range Assay. Only one plate is setup per six compounds in duplicate. Chemicals are added on top of plating media to give 1X final dilutions.

Figure 6. Suggested plate setup for Receptor Activity. Six plates are setup per six compound set in triplicate wells, duplicate plates (two sample test chemicals and controls per plate). Chemicals are added on top of plating media to give 1X final dilutions.

Green (wells H1-H6) = Positive Control

(50 nM β -estradiol or DHT)

Yellow (wells H7-H12) = Vehicle Control

(1% DMSO)

Other (wells A1-H11) = Sample Test Chemicals -

range 100 µM (row A) to 0.1 nM (row G) at

1:10 dilution in duplicate wells

Yellow (column 1) = Vehicle Control (1% DMSO)

Other (wells A2-F10) = Sample Test Chemicals – two

compounds per plate; range determined from

Dose Range plate at 1:5 dilutions in triplicate

wells (A2-C2 and D2-F2 for highest dose)

Green (wells G2-H10) = Positive Control at 1:5

dilutions (starting at 50 nM β-estradiol or DHT)

Dark Green (column 11) = Max Positive Control

(50 nM β-estradiol or DHT)

Purple (column 12) = Weak Response Control

(500 nM BPA or 125 nM progesterone)




A. Dose Range Assay (96-well plate)

Note: The Dose Range Assay must be performed PRIOR to starting the Receptor Activity Assay

1. Plate 8,000 cells per well of CryoRedi AR or ERα cells in 100 L plating medium following procedure outlined in

the Cell Preparation Section. Incubate overnight at 37 C, 5% CO2.

NOTE: To reduce variation between wells when using multiple plates, spread plates in the incubator (i.e., do not

stack plates).

2. Prepare stock solutions of compounds in sterile DMSO.

3. For Dose Range plate setup, we suggest that you make a master sample microplate containing ~490 µL Assay

Media (for Sample max concentration wells) and 450 µL DMSO Media (for Sample dilution wells). Prepare

Positive and Vehicle Control concentrations. Tables 2 and 3 give suggested examples for concentrations. Follow the

Plate map from Figure 5. Final starting concentrations for samples should be 100 µM (made up as 2x final

concentrations as 200 µM) with 1:10 dilutions in DMSO Media.

4. Add 100 μL of 2x concentrated working stock solution to the corresponding wells, including Positive and Vehicle

Controls.

5. Incubate plate for 20 hours (ERα) or 24 hours (AR) at 37 C in 5% CO2.

6. Prepare Fixation, Wash Buffer, and Staining Solution. Be sure that all solutions are at room temperature before use.

7. Remove plate(s) from incubator and carefully aspirate the media from the plate. Be sure to follow proper disposal

guidelines for appropriate compounds.

8. Add 100 μL of the Fixation Solution to each well.

9. Incubate the plate at room temperature for 20 minutes.

10. Carefully aspirate the fixation solution from the plate.

11. Carefully wash the plate twice with 1X Wash Buffer.

12. Aspirate buffer and replace with 100 μL/well of 1X Staining Solution.

13. Seal plate and incubate for at least 30 minutes at room temperature.

14. Perform image acquisition on the Thermo Scientific ToxInsight IVT Reader with proper protocol.

B. Receptor Activity Assay (per duplicate 96-well plates)

1. Plate 8,000 cells per well of CryoRedi AR or ERα cells in 100 L plating medium following procedure outlined in the Cell Preparation Section. We suggest that you pool the vials together to decrease subjective error for counting.

Incubate overnight at 37 C, 5% CO2.

2. Prepare Sample Working Stock solution. We suggest that you use a master sample microplate for compound

dilutions. The maximum final concentration for the Receptor Activity Assay will be determined from running the Dose Range Assay. Concentration should be calculated at 2x the final concentration (as it is being added on top of

the plating media in the wells). Remember that the DMSO concentration should remain the same. Concentration

should be calculated at 1 mL total volume per compound (for Sample max concentration wells) and 800 µL DMSO

Assay Media + 200 µL dilutions (1:5) as you will be doing triplicate wells in duplicate plates. In addition, prepare

Positive, Weak Response, and Vehicle Control concentrations.

a. For Activity Plate, follow plate map from Figure 6. Starting concentrations should be determined from

Dose Range Plate.

b. For Weak Response and Vehicle Control, only one concentration is used in 2 mL total volume. Please see

Tables 4 and 5 for example dilutions.

c. For Positive Control, note that you will be making up one concentration for column 11 (Max Positive) in

addition to using it as the starting concentration for the positive control dilution (suggestion is to make up

2.5 mL). Please see Tables 4 and 5 for actual dilutions.

3. Add 100 µL of 2x concentration working stock solution to corresponding wells, including Max Positive Control,

Positive Control dilutions, Weak Response Control, Vehicle Control, and Samples.

4. Procedure is followed identical to steps 4-14 in Section A for Dose Range Assay.




C. Data Analysis

*NOTE: The data acquired using the EPP Assay Cartridges should be analyzed with the provided Thermo

Scientific AddIn for Microsoft Excel and Excel Template for the EPP Assay for accurate prediction. Please see

the Assay Guide for more in-depth information on the templates.

1. The Thermo Scientific AddIn for Microsoft Excel and a copy of the EPP Assay Excel template are installed on the

computer connected to the ToxInsight instrument.

2. Open the Microsoft Excel 2010 software. On the Excel 2010 Ribbon, click the File tab and then click the New

option on the sidebar below the tab.

3. Click on the My Templates option under Available Templates.

4. A window with available templates will open. Click on the Thermo tab.

5. Select the appropriate template. The Excel template file will load. The .xltx extension indicates a template file.

a. For ERα Dose Range Assay, select EPP_DoseRange_ERa.xltx

b. For ERα Activity Assay, select EPP_ReceptorActivity_ERa.xltx

c. For AR Dose Range Assay, select EPP_DoseRange_AR.xltx

d. For AR Activity Assay, select EPP_ReceptorActivity_AR.xltx

6. Please follow instructions in the EPP Assay Guide and Excel AddIn User‟s Guide for additional information on

using the EPP Assay template and the Microsoft Excel AddIn tool. 7. Analyze the data according to the instructions.

D. Sample Results – ERα/AR Dose Range Assay

Figure 7 shows an example of the Graphs Tab for the Dose Range template with selected compounds graphed. User

defined thresholds allow you to set boundaries to determine if a compound has exceeded normalized controls. Note that the difference between ERα and AR Dose Range templates is that the AR template also graphs GFP translocation. All

compounds run can be viewed at the same time. The graphs represented are as follows:

1. Toxicity Index: a weighted measurement of #2-4

2. Object Count

3. Nuclear Intensity

4. Nuclear Size

5. Viability – focusing on Object Count

6. Foci Response: a combined measurement of #7-9

7. Foci Intensity

8. Foci Area

9. Foci Count

10. GFP Translocation (AR only)




Figure 7. Example of Graphs Tab in the ERα Dose Range Template

The Conclusions Tab of the Dose Range template shows the results for each individual compound by listing the

concentrations tested and indicates if each concentration exceeded the thresholds set in the Graphs Tab (Figure 8). A graph of the selected compound shows both toxicity and foci response along with a suggestion of the starting

concentration for the Activation Template. In addition, Plate information and Plate Acceptance criteria can be found to

ensure that your plates meet minimal data quality standards (please refer to the Assay Guide for additional details).

Figure 8. Example of Conclusions Tab in the ERα Dose Range Template




E. Sample Results – ERα/AR Receptor Activity Assay

Figure 9 shows an example of the Graphs Tab in the Receptor Activity Template. User defined thresholds determine if a

compound has exceeded normalized controls (for viability using object count) and thresholds set for each response

boundary. For example, one can determine if the compound has exceeded the Foci Response for the Weak Response

Control, and 20%, 50% and 100% of the Maximum Positive Response. In addition, you can determine if their

compounds are similar to their positive control (for ERα, with spot formation and degradation; with AR, spot formation and translocation). The graphs represented are as follows:

1. Viability – focusing on Object Count

2. Foci Response: a combined measurement of #3-5

3. Foci Intensity

4. Foci Area

5. Foci Count

6. GFP Translocation (AR only) or GFP Total Intensity (ERα only)

UniquePlateID Compound RepLine

ColorViability WR P1 P2 P3 P1 P2 P3

USPI2-SWTEST24_110520090002 b-estradiol Mean - + + + + + + +

USPI2-SWTEST24_110520090001 b-estradiol Mean - + + + + + + +

USPI2-SWTEST24_110520090002 compound A Mean - + + + + + - -

USPI2-SWTEST24_110520090001 compound A Mean - + + + + + - -

USPI2-SWTEST24_110520090002 compound B Mean - - - - - + - -

USPI2-SWTEST24_110520090001 compound B Mean - - - - - + - -

USPI2-SWTEST24_110520090001 compound B 3 - - - - - + - -

USPI2-SWTEST24_110520090001 compound B 1 - - - - - + + -

Foci Response GFP Nucleus Intensity

Threshold Threshold

Viability 80.00% P1 20.00%

P2 50.00%

P3 100.00%

SD

WR 1

Viability Foci Response

Figure 9. Example of Graphs Tab in the ERα Activity Template

The Conclusion Tab of the Receptor Activity template shows if the response of the selected compound exceeds the set

thresholds, determines the potency and magnitude of the response for each compound compared to the positive control,

and shows if the compounds mimic the positive control in respect to both foci response with degradation (ERα) or

translocation (AR). Multiple plates (up to 3) can be analyzed and graphed on the same template to ensure the validity of the results and monitor plate-to-plate variability. Figure 10 represents two separate plates of two different compounds

with β-estradiol as the positive control.




Foci Response %

UniquePlateID Compound Viability P1 WR P1 P2 P3 % Foci Response Line

USPI2-SWTEST24_110520090002 b-estradiol - + Mimic 0.00006 0.00008 0.00024 0.00041 109.90%

USPI2-SWTEST24_110520090001 b-estradiol - + Mimic 0.00005 0.00008 0.00028 0.00099 113.02%

USPI2-SWTEST24_110520090002 compound A - + No Mimic 0.31632 0.41951 0.69481 0.97012 192.23%

USPI2-SWTEST24_110520090001 compound A - + No Mimic 0.21476 0.33232 0.68140 1.00404 172.61%

USPI2-SWTEST24_110520090002 compound B - - No Mimic #N/A #N/A #N/A #N/A 11.02%

USPI2-SWTEST24_110520090001 compound B - - No Mimic 9.25110 #N/A #N/A #N/A 14.12%

Potential

Mimic

Figure 10. Example of Conclusions Tab in the ERα Activity Template

Recommendations for Automation

Plating Cells: To improve the uniformity and throughput of plating cells, use a liquid handling system such as a Thermo

Scientific Multidrop Combi or WellMate Dispensers.

Dead Volumes: Every piece of automation instrumentation has a non-recoverable dead volume associated with it. Be

aware of these dead volumes, priming volumes, and rinsing volumes when calculating your reagent requirements.

Nonspecific Binding: Because of the potential of reagent interaction with large surface areas inherent to tubing,

syringes, and peristaltic pumps, pre-priming with reagents or pre-coating with protein blockers may be warranted.

Mixing: Gentle mixing may be required when adding a DMSO-based solution to keep overly concentrated solutions

from lying on top of the cell layer. Be careful not to dislodge cells during mixing procedures.

Cell Washing: Use an automated plate washer designed to gently wash attached cells. Be careful not to dislodge cells

during cell washing.

Incubation: Minimize the time when plates with live cells are out of a controlled CO2 environment. For best results, use

an automated incubator to deliver plates to a pipetting deck.

Exposure: Some reagents and compounds are light-sensitive; be aware of these constraints when scaling up for an

automated run.

For the EPP Assays, two protocols are provided for each assay and each cell line. These protocols are available in the

Protocol Manager CompartmentalAnalysis.V4 folder of the ToxInsight IVT Platform Scan software. The protocols are as follows:

1. EPP_ERa_U2OS_CryoRedi_8000_20h_DoseRange_NuncEdge

2. EPP_AR_U2OS_CryoRedi_8000_24h_DoseRange_NuncEdge

3. EPP_ERa_U2OS_CryoRedi_8000_20h_Activity_NuncEdge

4. EPP_AR_U2OS_CryoRedi_8000_24h_Activity_NuncEdge

References

1. Levin, E. R. 2005. Molecular Endocrinology 19(8):1951-1959.

2. McEwan, I. J. 2004. Endocrine-Related Cancer 11, 281-293. 3. Rosenfeld, M. G., and C. K. Glass. 2001. J. Biol. Chem. 276, 36865-36868. 4. Mainwaring, W. I. 1977. Monogr. Endocrinol. 10, 1-178. 5. EPA. 2009. Endocrine Disruptor Screening Program Test Guidelines. OPPTS 890:1300: Estrogen Receptor Transcriptional

Activation (Human Cell Line (HeLa-9903)). EPA 740-C-09-006. Washington, DC: Office of Prevention, Pesticides and Toxic Substances, U.S. Environmental Protection Agency.




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