Frequently Asked Questions

The equipment and supplies needed to utilize INDIGO’s assay kits are
standard in most labs. Specifically, the materials you will need to
provide include:

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 and 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)
• Optional: antagonist reference compound
• Optional: clear 96-well assay plate, sterile, cell culture treated,
  for viewing cells on Day 2

Day 2

• Plate-reading luminometer

INDIGO’s kits are intended for research purposes only, and not for
diagnostic or therapeutic use in humans.

INDIGO’s 1×96-well assay kits contain materials to perform assays in a single 96-well assay plate. Our 3×32-well assay kits contain materials to perform three distinct groups of assays in a 96-well plate format. Reagents are configured so that each group will comprise 32 assays. If desired, however, reagents may be combined to perform either 64 or 96 assays. Please note, the aliquot of 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.

2-fold is what we have found to be historically, mathematically significant.

A 15% drop in live cells is considered statistically significant.

The compound screening medium (CSM) included in nearly all INDIGO kits contains 10% charcoal-stripped fetal bovine serum (FBS). (The exceptions to this are our RORg assays(5%) and TGF-beta (2%).) If your test substance is human serum, we do recommend serum-free medium, which is available at no charge but must be requested at the time of order.

INDIGO’s assays are luminescence-based, not fluorescence-based. Therefore, one does not designate specific wavelengths (or filters of any kind) when reading in luminescence mode. Total photon emission is quantified.

You always want to add the compounds to the cells, rather than adding the
cells to the compounds, per the assay protocol.

The concentration of organic solvent carried over into the assay wells
should not exceed 0.1%. We recommend no more than 0.4% DMSO carry-over
into the assay wells.

It is okay to thaw and keep the CSM/CRM refrigerated between uses. However, we recommend it only be stored at 4°C for a week or less, and that for any time longer than that, that it be refrozen.

Yes, we recommend keeping it at -20C.

Yes, you can thaw the LCMA buffer and keep it at 4C. You can freeze/thaw
the LCMA substrate up to three times.

We do not recommend Tryptan Blue for checking cell viability, as it can be inaccurate for recently thawed cells that are recovering from the trauma
of thawing.

All of INDIGO’s assay kits are shipped on dry ice. For customers in the contiguous United States, all orders are shipped via overnight delivery. For customers in the United Kingdom, orders are shipped via priority delivery services and should reach your location within 3-4 days. For customers outside the US and UK, please visit our Distributors Page to find which of our trusted Distributors to contact with your order. 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. The date of product expiration is printed on the Product Qualification Insert (PQI) enclosed with each kit.

INDIGO offers an ongoing kit promotion throughout the year: for every ten assay kits purchased (in a single order), customers receive an additional assay kit free of charge. For orders of 30 or more kits, please contact INDIGO’s customer service team to discuss potential volume sales opportunities. For customers purchasing assay kits that are looking to run smaller studies, purchasing a 3×32-format kit may provide some additional savings. See the information below or contact INDIGO’s team for more information.

If you are looking for an initial go/no-go response, we recommend 1-3 dose concentrations. For EC50/IC50 values, we recommend at least 7-8, with 7 being the absolute minimum. You are welcome to do more dose concentrations if preferred, and we find that many of our customers opt to do 10 doses for a full EC50/IC50 curve.

Treatment concentrations are prepared at INDIGO using serial dilutions in fixed increments; the starting concentration and increment of dilution is specified by the customer via our Study Work Order sheet that accompanies all screening studies. For accurate determination of EC50 and/or IC50 values, we recommend at minimum spanning a 5,000-fold concentration range over 8 doses. This strategy requires a 3.33-fold or 4-fold increment of serial dilution. The minimum doses recommended for EC50 and/or IC50 value is 7 test concentrations.

You can design your study to include as many replicates as you would like. If you are looking to utilize your data to publish, at least three replicates are recommended.

While you are certainly welcome to run the LCMA with agonist mode, we do not typically recommend needing to do so. The reason for this is because cytotoxicity would produce a downward curve. In agonist mode, an agonist response creates an upwards curve and if there are any cytotoxic effects they typically occur towards the top of the curve at the highest doses. Therefore, you would see the downward turn at those levels. In antagonist or inverse-agonist mode, the curve is downward to begin with. In this case, the LCMA is recommended to confirm if the downward curve is a true response or due to cytotoxic effects of the treatment compound.

If test samples are provided as concentrated stock solution, we prefer a 1000x concentration stock relative to the highest treatment concentration (e.g., provide > 10mM stocks if the highest assay concentration is 10 uM). This ensures that the concentration of organic solvent carried over into the assay wells is ~0.1%. INDIGO will not perform studies that require greater than 0.4% DMSO carry-over into the assay wells. You will also need to disclose the solvent used to generate the Master Stock (DMSO preferred—please inquire about the use of alternative organic solvents). Please provide approximately 50uL of 1000x concentrated stock solutions. If test samples are provided in powder form, they must be pre-weighed by the client. INDIGO does not perform manual weighing of test materials. Please provide no more than 5 mg of dry material. You will also need to provide the molecular weight (MW; g/mol) of each test compound. If test samples are provided as neat liquids, provide the Molar concentration of
the liquid or provide a pre-weighed volume of the liquid sample (e.g., x mg/100ul). You will also need to provide the molecular weight (MW; g/mol) of each test compound. Aqueous solutions may be submitted, but they must be filter-sterilized and devoid of preservatives and chelating agents. Additional charges will be applied if extensive sample handling is required to generate “assay-ready” stocks.

INDIGO retains compounds for three months after reporting results, then disposes of or returns the compounds. If you want us to return your compounds or to store them for more than three months, you must contact us to make appropriate arrangements prior to the three-month window expiring. We reserve the right to charge a disposal fee. We may charge a monthly fee for long-term storage. We will store all compounds under locked, temperature-controlled conditions. An internal chain of custody is maintained for each lot of compound received. Additional information relating to product and service agreements can be found in our Terms and Conditions.

Upon completion of your study, you will receive a detailed study report (PDF format) including all assay methods and validation, the raw data and calculations of your study (Excel format), and graphing files of all data (GraphPad Prism format). In addition, you are always welcome to contact INDIGO’s team for assistance in interpretation of your data.

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.

View our list of reference compounds. 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.