Frequently Asked Questions

INDIGO’s assays are not binding assays. They are cell-based trans-activation assays, and the principal application is in the screening of test samples to quantify any functional activity, either agonist or antagonist, that the compounds may exert against the nuclear receptors. INDIGO reporter systems utilize firefly luciferase reporter gene technology, and while there is a binding taking place, our assays do not measure it. Instead, the luciferase light response is measured which correlates to the activation status of the receptor (either activation or inhibition).

Quantifying changes in luciferase expression in the treated reporter cells provides a sensitive surrogate measure of the changes in receptor activity. It will tell the scientist the significance (strength) of the interaction by the level of light emitted. In addition, cell-based assays are more sensitive and able to detect smaller levels of activation.

INDIGO’s nuclear receptor assays utilize proprietary human and non-human mammalian cells engineered to provide constitutive, high-level expression of the designated receptor. Specific cell type information for each assay is proprietary and available only through consultation with INDIGO’s technical team following a screening service or assay kit purchase.

Reporter cells included in INIDIGO’s steroid hormone nuclear assay kits (ERα, ERβ, AR, PGR, MR, GR) express the native, full-length receptors. INDIGO’s other nuclear receptor assays, however, include reporter cells that express hybrid nuclear receptors. In these cases, the respective receptor’s native N-terminal sequence comprising the DNA Binding Domain (DBD) has been replaced with sequence encoding the yeast Gal4-DBD. All other native NR functional/structural domains (ligand binding domain, hinge region, and various activation domains) are present in these hybrid receptors.

These reporter cells also contain the firefly luciferase reporter gene functionally linked to the upstream genetic response element for Gal4. Consequently, once a bioactive compound associates with the ligand binding domain of the hybrid receptor, only the luciferase reporter gene is induced. Ligand-activation of the hybrid receptor will not induce collateral expression of target genes that are otherwise regulated by the native nuclear receptor.

We do utilize commercially available reference agonists for our assays, and this reference agonist is included in each assay kit. We do use reference antagonists where a reference is known and commercially available. Some of our antagonist assays do not have reference antagonists available.

Normalizing RLU data to cell numbers or protein content is not something we do or recommend. The suspension of reporter cells is a ‘master reagent’ that should be dispensed with high precision into all wells of the assay plate. Any significant downstream variability in cell number or health will be the result of cytotoxicity induced by the test compound treatments. It is certainly important to perform cytotoxicity analyses for antagonist-mode assays (and inverse-agonist mode assays) to identify and weed out false-positive data, and INDIGO’s Live Cell Multiplex (LCM) Assay works very well for that purpose. However, attempting to normalize RLU data from treated cells that are in metabolic crisis to untreated, healthy control cells can result in gross misinterpretations of the data.

When performing cell-based trans-activation assays (which rely on the coordinated cell-cycle processes of gene induction, transcription, translation, mRNA and protein turn-over) “healthy cells” and “dying cells” are not equal units, so attempting to normalize assay data to any cell-related endpoint is not a sound method.

Several nuclear receptors express high-level constitutive activity that is (seemingly) independent of a ligand interaction. The RORs, the ERRs, CAR-1, and LRH-1 are nuclear receptors with this activity profile, and they are loosely referred to as “inverse-agonist receptors/assays.” In practice, however, when a bio-active compound associates with the receptor’s ligand binding domain, there will be one of two functional outcomes: 1.) There will be a dose-dependent decrease in the receptor’s constitutive activity (an inverse-agonist response), or 2.) there will be a moderate dose-dependent increase in activity above the receptor’s already high constitutive level (an agonist response).

INDIGO’s assays are capable of showing both of these alternative response in a single conventional assay setup.

Yes. When one is looking for agonist responses exclusively it is a common strategy to suppress the constitutive activity of the receptor by co-treating with a fixed concentration of the inverse-agonist reference compound provided with the assay kit. For example, this strategy is frequently used by researchers screening for agonists of RORγ. RORγ reporter cells are co-treated with a fixed concentration of Ursolic Acid (INDIGO’s reference inverse-agonist) and varying concentrations of the user’s test compounds. Compounds that are putative agonists of RORγ will show a significant dose-dependent increase in RORγ activity above the suppressed level.

First, it is important to understand the basic molecular biology behind the RORγ and RORγt isoforms. In humans, RORγ and RORγt derive from the same gene. However, expression of the respective transcripts is driven by tissue-specific alternative promoters, resulting in transcripts with variant exon 1 sequences. Consequently, the translated proteins have unique N-terminal amino acid sequences that encode unique DNA binding domains (DBD) for RORγ and RORγt. Importantly, the RORγ and -γt isoforms have common Ligand Binding Domains, as well as all other functional/structural domains.

Therefore, it is their differential expression between tissues and cell types, and their variant DBD sequences, that account for their different physiological activities (target gene expression profiles). INDIGO’s RORγ reporter cells express an engineered cDNA that encodes a hybrid receptor. Specifically, the N-terminal amino acid sequence encoding either the RORγ DBD or the RORγt DBD has been replaced with amino acid sequence encoding the yeast GAL4-DBD. Hence, INDIGO’s reporter cells express a Gal4(DBD)-RORγ hybrid receptor—a “generic RORγ”—that is blind to the isoform differences in function and target gene expression profiles that otherwise exist in vivo.

The specific utility of INDIGO’s RORγ assay is to quantify and rank interactions that occur between test compounds and the RORγ LBD. These reporter cells will show RORγ inverse-agonist and agonist responses that may be induced through drug interactions with the receptor.

Yes. Preferably the extracts will be aqueous preparations that are: 1.) homogenous solutions with no particulate material, and 2.) sterile. If either is not true, the sample should be passed through a 0.2um syringe-tip filter. Preferably aqueous preparations will be no less than 4x-concentrated relative to the final treatment concentration in the assay. If the extract is prepared in DMSO, a stock of no less than 250x-concentrated (preferably more concentrated) is required. Residual DMSO carried over into any of INDIGO’s nuclear receptor assays should never exceed 0.4%. Higher DMSO concentrations will degrade assay performance.

"First, it is important to understand that EC50 (and IC50) values are not constants. There are numerous variables that impact these, and other, assay metrics. Consequently, these values can vary greatly between different assay types and between different related receptors. Regarding this last point: Neither ERα and ERβ, or TRα and TRβ, are simple iso-forms of each other; rather, they are all distinct receptors encoded by separate genes. As an example, the two ER proteins share only 45% sequence identity.

If one includes ‘conserved’ substitutions between the two amino acid sequences, the two receptors still display only 60% sequence homology. While the two ERs have evolved to bind the physiological activator 17β-estradiol, it would be quite surprising if these two different nuclear receptors had the same activation profiles within the cellular context. It is also worth noting that the two ERs display strikingly disparate ligand preferences and activation/inhibition profiles for many other bioactive chemicals.

INDIGO’s nuclear receptor assays are cell-based trans-activation assays, which rely on a complex orchestration of the many molecular processes that occur in vivo. In brief, these include: the reporter cells take up exogenous drug, which also experiences competitive interactions with serum-based and cellular macromolecules > binding of the drug to the ligand-binding domain of the nuclear receptor, which initiates the shedding of co-repressor molecules and recruitment of co-activator molecules > nuclear-translocation > binding of the activated receptor complex to the genetic response elements of the gene promoter > formation of a complete transcription complex > induced expression of the target gene (in our case, Luciferase) > translation of mRNA.

The mechanics of such cell-based trans-activation assays are in sharp contrast to in vitro ligand-binding assays and/or co-factor recruitment assays. Such assays measure a simple A+B → AB interaction between two purified molecules, typically polypeptides corresponding to the (much more highly conserved) ligand-binding domain sequences of the respective receptors, or to very small polypeptides corresponding to a specific protein-protein interaction sequence of a co-activator protein.

While useful for some applications, these various in vitro assay formats do not represent the complex molecular interactions that occur between ligands and the native receptors within the cellular environment. Therefore, when comparing the data sets from all of these various assay types, one can expect to see widely varying assay metrics, including respective EC50/IC50 values."

Yes. INDIGO’s Aryl Hydrocarbon Receptor (AhR) and Estrogen Receptor Alpha (ERα) bioassay have been approved by the California Water Boards, State Water Resources Control Board, for use as bioanalytical screening tools for Constituents of Emerging Concern (CECs) in recycled water.