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Human HNF4a Reporter Assay Kit

SIZE SKU PRICE
1 x-96 well format assays
1 x-384 well format assays
SIZE SKU
1 x-384 well format assays
1 x-96 well format assays

Product Description and Product Data

This is an all-inclusive cell-based luciferase reporter assay kit targeting the Human Hepatocyte Nuclear Factor 4 alpha (NR2A1, HNF4a). INDIGO’s HNF4a reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of the HNF4a. In addition to HNF4a 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. The principal application of this assay is in the screening of test samples to quantify any functional activity, either agonist or antagonist, that they may exert against HNF4a. This kit provides researchers with clear, reproducible results, exceptional cell viability post-thaw, and consistent results lot to lot. Kits must be stored at -80C. Do not store in liquid nitrogen. Note: reporter cells cannot be refrozen or maintained in extended culture.

Features

  • Clear, Reproducible Results

  • All-Inclusive Assay Systems
  • Exceptional Cell Viability Post-Thaw
  • Consistent Results Lot to Lot

Product Specifications

Target TypeNuclear Hormone Receptor
SpeciesHuman
Receptor FormHybrid
Assay ModeAgonist, Inverse Agonist
Kit Components
  • Human HNF4a Reporter Cells
  • Cell Recovery Medium (CRM)
  • Compound Screening Medium (CSM)
  • CCBA, (ref. agonist; in DMSO)
  • Detection Substrate
  • Detection Buffer
  • White, sterile, cell-culture ready assay plate
Shelf Life6 months
Shipping RequirementsDry Ice
Storage temperature-80C

Data

Agonist dose-response of Human HNF4a. Agonist dose-response of Human HNF4a reporter cells using CCBA (provided) and Alverine Citrate (Millipore Sigma). Luminescence was quantified and average relative light units (RLU) and their corresponding values of standard deviation (SD) and Fold- Activation were determined for each treatment group (n = 3). GraphPad Prism software was used to curve-fit data using the least-squares method of non-linear regression, and EC50 values were determined.
Inverse-Agonist dose-response of Human HNF4a. Inverse-agonist response of HNF4a reporter cells using BI60153 (Cayman Chem.). Luminescence was quantified and average relative light units (RLU) and their corresponding values of standard deviation (SD) and Fold-Reduction were determined for each treatment group (n = 3). INDIGO’s Live Cell Multiplex assay was performed and confirmed the absence of compound-induced cytotoxicity (data not shown).

Target Background

This nuclear receptor assay utilizes proprietary human cells engineered to provide constitutive, high-level expression of the Human Hepatocyte Nuclear Factor 4 alpha (NR2A1), a ligand-dependent transcription factor known as HNF4a.

In INDIGO’s Reporter Cells the HNF4a receptor is expressed as a hybrid protein in which its native DNA binding domain (DBD) sequence has been exchanged with that of the DBD sequence from the yeast Gal4 gene. Additionally, the cells include the luciferase reporter gene functionally linked to the Gal4 upstream activation sequence (UAS) and a minimal promoter. HNF4a undergoes nuclear translocation, DNA binding, recruitment and assembly of the co-activators and accessory factors required to form a functional transcription complex, culminating in expression of the reporter gene. Thus, quantifying changes in luciferase expression in the treated reporter cells provides a specific and sensitive surrogate measure of the changes in HNF4a activity. Unlike some other assay formats, the readout from INDIGO’s reporter cells demands the same orchestration of all intracellular molecular interactions and events that can be expected to occur in vivo.

As is true in vivo, the expressed hybrid HNF4a receptor displays a moderate level of constitutively activity. A true ligand interaction may provoke either i.) an agonist response that delivers an additional increase in receptor activity above its already high constitutive level of activity, or ii.) an inverse-agonist response in which a loss of receptor activity is observed. The principal application of this assay is in the screening of test compounds to quantify inverse-agonist or agonist activities that they may exert against the Human HNF4a receptor.

These HNF4a Reporter Cells are prepared using INDIGO’s proprietary CryoMite™ process. This cryo-preservation method yields high 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, or cell titer adjustments prior to assay setup.

Also available as a service

Hepatocyte Nuclear Factor 4 alpha (HNFa, NR2A1)

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