Frequently Asked Questions
Find answers to questions we frequently receive regarding our assay kits and services.
Each kit includes luciferase reporter cells, an assay plate, all required reagents (optimized culture media, positive-control agonist, and detection reagent), and a detailed Technical Manual. A luminometer is required for Relative Light Unit (RLU) measurements.
INDIGO's Nuclear Receptor Assay Kits use transiently transfected cells, less prone to genetic drift and batch-to-batch variations. Moreover, they do not require prolonged cell culture, significantly reducing time and contamination risks. Simply thaw the reporter cells, and you are ready to perform the assay. Contact us to learn how our assay kits can fit into your workflow.
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 one month 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 one month, you must contact us to make appropriate arrangements prior to the one-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.