Do I need any special equipment to utilize INDIGO’s assay kits?
standard in most labs. Specifically, the materials you will need to
- 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
- 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
- Plate-reading luminometer
Can INDIGO’s Nuclear Receptor Assay Kits be used for diagnostic purposes?
diagnostic or therapeutic use in humans.
What is the difference between the 1×96-well and 3×32 assays in a 96-well plate formats?
Do you have a specific value for positive nuclear receptor activations for your assay kits?
Do INDIGO kits use Fetal Bovine Serum?
Which wavelength should I use to read my plate?
Is it okay to plate the test compounds into the wells then immediately add the cells after they have been properly reconstituted?
cells to the compounds, per the assay protocol.
What concentration of organic solvent carried over into the assay wells is acceptable for INDIGO reporter assays?
should not exceed 0.1%. We recommend no more than 0.4% DMSO carry-over
into the assay wells.
Can compound screening medium (CSM) and cell recovery medium (CRM) be reused after the initial thaw? (3×32-well assay kit-specific)
Can I thaw and refreeze the 4mM Staurosporine provided in your LCMA kits?
Can I thaw and refreeze LCMA buffer and LCMA substrate?
the LCMA substrate up to three times.
Can Tryptan Blue be used to check viability (QA) before running the assays?
How are your products shipped?
Are there any discounts or special promotions?
How many dose concentrations do you recommend?
Can I choose the exact concentrations I want my compounds to be screened at?
How many replicates do you recommend?
Why do you recommend running the LCMA for antagonist and inverse-agonist modes, but not agonist mode?
How much of my compound should I send for a screening study?
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.
How long does INDIGO retain client compounds once a study has been completed?
What kind of data will I receive from my screening study?
Are INDIGO assays binding assays, or does INDIGO offer binding assays?
What cell lines/types are used?
Do INDIGO’s nuclear receptor assays express the native, full-length receptor?
Can you tell me what the reference agonist/antagonist is for your receptor assays?
What is the best way to normalize data (e.g., cell number, protein content)?
What is an inverse-agonist nuclear receptor assay? Are these assays run separately in “agonist” and “inverse-agonist” mode?
Can the INDIGO’s ‘inverse-agonist’ receptor assays (RORα, RORγ, ERRα, ERRγ, CAR-1, and LRH-1) be set up to suppress the high constitutive activity of the receptor, thereby allowing the assessment of agonist activities?
Do INDIGO’s RORγ reporter cells express the RORγt isoform of the receptor?
Can INDIGO’s nuclear receptor assays be used to test processed extracts derived from waste-water treatment samples, environmental samples, plant samples, etc.?
Why don’t INDIGO’s ERα and ERβ assays produce the same EC50 values for 17β-estradiol? Likewise, for your TRα and TRβ assays run against triiodothyronine? There are reports that the respective EC50 values should be the same for these related pairs of nuclear receptors.
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.