|Description:||This nuclear receptor assay system utilizes proprietary human cells engineered to provide constitutive, high-level expression of the human thyroid hormone receptor beta (NR1A2), a ligand-dependent transcription factor commonly referred to as TRβ. The reporter cells include the luciferase reporter gene functionally linked to a TRβ-responsive promoter. Thus, quantifying changes in luciferase expression in the treated reporter cells provides a sensitive surrogate measure of the changes in TRβ activity. The principal application of this reporter assay system is in the screening of test samples to quantify any functional activity, either agonist or antagonist, that they may exert against human TRβ.
TRβ reporter cells are prepared using the proprietary CryoMite process. This cryo-preservation method yields exceptional cell viability post-thaw, and provides the convenience of immediately dispensing healthy, division-competent reporter cells into assay plates. There is no need for cumbersome intermediate treatment steps such as spin-and-rinse of cells, viability determinations, cell titer adjustments, or the pre-incubation of reporter cells prior to assay setup.
The nuclear receptor reporter assays are all-inclusive cell-based assay systems. In addition to TRβ reporter cells, this kit provides two optimized media for use during cell culture and in diluting the user’s test samples, a reference agonist, luciferase detection reagent, and a cell culture-ready assay plate.
|Stability:||≥ 6 months|
|Storage:||The aliquot of TRβ Reporter Cells is provided as a single-use reagent. Once thawed, reporter cells can NOT be refrozen or maintained in extended culture with any hope of retaining downstream assay performance. Therefore, extra volumes of these reagents should be discarded after assay setup.
Assay kits are shipped on dry ice. Upon receipt, individual kit components may be stored at the temperatures indicated on their respective labels. Alternatively, the entire kit may be further stored at -80°C.
To ensure maximal viability, “Reporter Cells” must be maintained at -80°C until immediately prior to use.
|Synonyms:||TRβ (human) Reporter; TRβ|
|Kit Components:||▪ TRβ Reporter Cells: 1 x 2.0 mL; -80°C
▪ Cell Recovery Medium (CRM): 2 x 10.5 mL; -20°C
▪ Compound Screening Medium (CSM): 1 x 45 mL; -20°C
▪ L-Triiodothyronine (150 uM in DMSO) (reference agonist for TR's): 1 x 30 µL; -20°C
▪ Detection Substrate: 1 x 6.0 mL; -80°C
▪ Detection Buffer: 1 x 6.0 mL; -20°C
▪ 96-well collagen-coated assay plate (white, sterile, cell-culture ready): 1; -20°C
|Materials Required but Not Supplied:||The following materials must be provided by the user, and should be made ready prior to initiating the assay procedure:
DAY 1 ▪ cell culture-rated laminar flow hood.
▪ 37°C, humidified 5% CO2 incubator for mammalian cell culture.
▪ 37°C water bath.
▪ 70% alcohol wipes
▪ 8- or 12-channel electronic, repeat-dispensing pipettes & sterile tips
▪ disposable media basins, sterile.
▪ sterile multi-channel media basins (such as the Heathrow Scientific "Dual- Function Solution Basin"), or deep-well plates, or appropriate similar vessel for generating dilution series of reference compound(s) and test compound(s).
▪ antagonist reference compound (optional).
DAY 2 ▪ plate-reading luminometer.
|Preparation:||Most commonly, test compounds are solvated at high-concentration in DMSO, and these are stored as master stocks. Master stocks are then diluted to appropriate working concentrations immediately prior to setting up the assay. Users are advised to dilute test compounds to 2x concentration stocks using Compound Screening Medium (CSM), as described in Step 2 of the Assay Protocol. This method avoids the adverse effects of introducing high concentrations of DMSO into the assay. The final concentration of total DMSO carried over into assay reactions should never exceed 0.4%.
NOTE: CSM is formulated to help stabilize hydrophobic test compounds in the aqueous environment of the assay mixture. Nonetheless, high concentrations of extremely hydrophobic test compounds diluted in CSM may lack long-term stability and/or solubility, especially if further stored at low temperatures. Hence, it is recommended that test compound dilutions are prepared in CSM immediately prior to assay setup, and are considered to be 'single-use' reagents.
|Assay Protocol:||Review the entire Assay Protocol before starting. Completing the assay requires an overnight incubation. Steps 1-8 are performed on Day 1, requiring less than 2 hours to complete. Steps 9-15 are performed on Day 2, and require less than 1 hour to complete.
▪ A word about Antagonist-mode assay setup ▪
Receptor inhibition assays expose the Reporter Cells to a constant, sub-maximal concentration (typically between EC50 – EC85) of a known agonist AND the test compound(s) to be evaluated for antagonist activity. This TRβ Reporter Assay System kit includes a 2.4 mM stock solution of L-Triiodothyronine, an agonist of TRβ that may be used to setup antagonist-mode assays. 200 nM L-Triiodothyronine typically approximates EC80 in this reporter assay. Hence, it presents a reasonable assay concentration of agonist to be used when screening test compounds for inhibitory activity.
We find that adding the reference agonist to the bulk suspension of Reporter Cells (i.e., prior to dispensing into assay wells) is the most efficient and precise method of setting up antagonist assays, and it is the method presented in Step 5b of the following protocol. Note that, in Step 6, 100 µl of treatment media is combined with 100 µl of pre-dispensed [Reporter Cells + agonist]. Consequently, one must prepare the bulk suspension of Reporter Cells to contain a 2x-concentration of the reference agonist.
DAY 1 Assay Protocol: All steps must be performed using proper aseptic technique.
1) Remove Cell Recovery Medium (CRM) and Compound Screening Medium (CSM) from freezer storage and thaw.
▪ CRM should be thawed and equilibrated to 37°C using a water bath. CRM pre-warmed to 37°C is required in Step 3.
▪ CSM may be thawed in a 37°C water bath.
2.) Prepare Test Compound(s) and Reference Compound stocks to be screened for Agonist or Antagonist activities.
The final concentration of total DMSO carried over into assay reactions should never exceed 0.4%.
Note that, in Step 6, 100 µl of the prepared treatment media is added into assay wells that have been pre-dispensed with 100 µl of Reporter Cells. Hence, to achieve the desired final assay concentrations one must prepare treatment media with a 2xconcentration of the test and reference material(s). Use CSM to prepare the appropriate dilution series. Plan dilution schemes carefully. This assay kit provides 35 ml of CSM.
This TRβ Reporter Assay System kit includes a 2.4 mM stock solution of LTriiodothyronine, a reference agonist of TRβ. We find the following 7-point treatment series, with concentrations presented in 3-fold decrements, provides a suitable dose-response: 2,400, 800, 267, 88.9, 29.6, 9.88, and 3.29 nM, and including a 'no treatment' control.
3.) First, retrieve the tube of CRM from the 37°C water bath and sanitize the outside with a 70% ethanol swab.
Second, retrieve Reporter Cells from -80°C storage. Perform a rapid thaw of the frozen cells by transferring a 10.0 ml volume of 37°C CRM into the tube of frozen cells. Recap the tube of Reporter Cells and immediately place it in a 37°C water bath for 5 - 10 minutes. The resulting volume of cell suspension will be 12 ml.
Third, work in the cell culture hood to carefully mount four sterile 8-well strips into the blank assay plate frame. Strip-wells are fragile. Note that they have keyed ends (square and round), hence, they will fit into the plate frame in only one orientation.
4.) Retrieve the tube of Reporter Cell Suspension from the water bath. Sanitize the outside surface of the tube with a 70% alcohol swab.
5.) a. Agonist-mode assays. Invert the tube of Reporter Cells several times to disperse cell aggregates and gain an homogenous cell suspension. Without delay, dispense 100 µl of cell suspension into each well of the 96-well Assay Plate.
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b. Antagonist-mode assays. Invert the tube of Reporter Cells several times to disperse any cell aggregates, and to gain an homogenous cell suspension. Supplement the bulk suspension of Reporter Cells with the desired 2x-concentration of reference agonist. Dispense 100 µl of cell suspension into each well of the 96-well Assay Plate.
NOTE 5.1: Take special care to prevent cells from settling during the dispensing period. Allowing cells to settle during the transfer process, and/or lack of precision in dispensing uniform volumes across the assay plate will cause wellto-well variation (= increased Standard Deviation) in the assay.
NOTE 5.2: Users sometimes prefer to examine the reporter cells using a microscope. If so, the extra volume of cell suspension provided with each kit may be dispensed into a clear 96-well plate, treated +/- test compounds as desired, and incubated overnight in identical manner to those reporter cells contained in the white assay plate.
6.) Dispense 100 µl of 2x-concentration treatment media (prepared as described in Step 2) into appropriate wells of the assay plate.
7.) Transfer the assay plate into a 37°C, humidified 5% CO2 incubator for 22 - 24 hours.
NOTE: Ensure a high-humidity (> 90%) environment within the cell culture incubator. This is critical to prevent the onset of deleterious "edge-effects" in the assay plate.
8.) For greater convenience on Day 2, retrieve Detection Substrate and Detection Buffer from -80°C storage and place them in a dark refrigerator (4°C) to thaw overnight.
DAY 2 Assay Protocol: Subsequent manipulations do not require special regard for aseptic technique, and may be performed on a bench top.
9.) 30 minutes before intending to quantify TRβ activity, remove Detection Substrate and Detection Buffer from the refrigerator and place them in a low-light area so that they may equilibrate to room temperature. Once at room temperature, gently invert each tube several times to ensure homogenous solutions.
NOTE: Do NOT actively warm Detection Substrate above room temperature. If these solutions were not allowed to thaw overnight at 4°C, a room temperature water bath may be used to expedite thawing.
10.) Turn on the luminometer. Set the instrument to perform a single 5 second “plate shake” prior to reading the first assay well. Read time may be 0.5 second (500 mSec) per well, or less.
11.) Immediately before proceeding to Step 12, transfer the entire volume of Detection Buffer into the vial of Detection Substrate, thereby generating a 12 ml volume of Luciferase Detection Reagent (LDR). Mix gently to avoid foaming.
12.) Following 22 - 24 hours of incubation, retrieve the assay plate from the incubator. Remove the plate’s lid and discard all media contents by ejecting it into an appropriate waste container. Gently tap the inverted plate onto a clean absorbent paper towel to remove residual droplets. Cells will remain tightly adhered to well bottoms.
13.) Add 100 µl per well of LDR to the assay plate.
14) Allow the assay plate to rest at room temperature for at least 5 minutes following the addition of LDR. Do not shake the assay plate during this period.
15) Between 5 - 90 minutes after adding LDR, place the assay plate in the luminometer and quantify luminescence.