Scientific Frequently Asked Questions

Finding Product Information

Where do I find my item’s specific plating density, incubation time, etc.?

The user manual is generic and applies to one or more products of a given class. For product-specific information like incubation conditions and cell plating density, refer to the product’s datasheet that is found on the product’s details webpage.

What cell plating time, agonist incubation time, and agonist incubation temperature should I use for

Without knowing the reference agonist, optimal assay conditions for orphan GPCR assays are unknown. However, we can suggest a starting point to use until an agonist is discovered that could later be used to optimize assay conditions. When working with a cell line, assays should be run the day after cells are plated. When working with an eXpress kit, cells may take as long as 48 hours to recover after they have been thawed and plated. Therefore, for eXpress assay cells (for orphan GPCRs), we recommend that cells be allowed to recover for 48 hours after they are thawed and plated. If an agonist is discovered, then it would be possible to test if a 24 hour cell recovery is sufficient. Ligand incubation periods should start at 90 minutes at 37°C. Agonist incubation periods should generally not be less than 90 minutes. If an agonist is discovered, then incubations longer than 90 minutes and/or room temperature incubations can be tested.

Is the receptor in my product full length?

The product datasheet will provide the accession number corresponding to the protein receptor’s sequence. If an amino acid range is provided, this indicates which amino acids were included. If no amino acid range is listed, then the full length of the receptor was used. Most Eurofins DiscoverX products use full-length receptors, with the exception of the dimerization cell lines that mostly use truncated receptors lacking the cytoplasmic tail.

Can I use a dimerization cell line to monitor downstream signaling?

Many dimerization cell lines express truncated receptors that lack the cytoplasmic tail and therefore will not transduce the ligand-induced signal any further than the dimerization event. Thus, attempting to measure a downstream signaling step will fail. In order to determine if the dimerization cell line you are interested in expresses full length receptors and would have intact downstream signaling, see the FAQ question “Is the receptor in my product full length?” above.

Where can I obtain safety information about my cell-based product?

For genetically modified organism (GMO) information on transgenes and engineering of a cell product, obtain the biosafety document from technical support with an ~3 day lead time.
For information on the viral-free status of Eurofins DiscoverX cell products that were made using Moloney Murine Leukemia Virus (MMLV) methods, obtain the marker rescue document. Request these from Technical Support (approx. 3 week lead time for cell growth)
For information on recommended US biosafety level of Eurofins DiscoverX products, please contact technical support.

Product Validation

How are the cells modified to express their transgenes?

Most Eurofins DiscoverX cell-based products use random genomic insertion mediated by a replication incompetent Moloney Murine Leukemia Virus (MMLV) to incorporate the transgene into the genome of the target cells. Some product lines (e.g., ChemiSCREEN™, ChemiBRITE™) use a transfection method to insert a plasmid that remains in the cells and from which the transgene is expressed. The PathHunter® Signaling Reporter and SPRINTer™ Targeted Protein Degradation assays use CRIPSR/Cas9 to insert the construct into the genome, and the components are all provided to the cells using a virus-free transfection method. In the case of the SPRINTer assays, the insertion is a precisely targeted knock-in to add the Enzyme Fragment Complementation (EFC) small enzyme donor (ED) tag in-frame to the end of the endogenous gene locus.

How were clones chosen? Is transgene expression measured by flow cytometry?

Typically, stable pools and clonal cell line candidates are evaluated for their functional response to a ligand, small molecule inhibitor, or large molecule with inhibitory or agonistic characteristics with IC/EC₅₀s and signal:background ratios being the typical performance-based metrics used. Cell assays are also evaluated for stability of their functional performance over 10-15 passages. However, there are some Eurofins DiscoverX cell lines from which expression data was generated via flow cytometry analysis. For example, some PathHunter® Checkpoint Receptor cell lines have been analyzed in this manner. On the other hand, GPCR cell lines have not been tested for the density of receptors at the cell membranes. It is typically impractical to run FACs analysis of GPCR cell lines due to the generally poor quality of anti-GPCR antibodies that are available.

How stable are the cell-based products?

Eurofins DiscoverX cell-based products are tested for at least 10 passages of consistent assay performance, but many products have actually been tested for at least 15 passages; some for many more than this. With the products that are made using genomic insertion of the transgenes (see ‘How are the cells modified to express their transgenes?’ question above for a list), the cells’ expression of the transgenes is consequently particularly stable and may persist for many passages even in absence of antibiotics (although this is not recommended practice). Though transfected cell products are not typically quite as stable and rely more on antibiotic selection, they have all been tested for stability through at least those 10 passages when kept under selection.

How are the GPCR orphan receptor products validated?

Since by definition, there are no agonists for orphan GPCR receptors, Eurofins DiscoverX uses two assays meant to establish that the transgenes are expressed and could, if an agonist was found, respond and produce a signal. The first one is a modified Western blot. Lysate from the cells is run through a gel and onto a membrane. The membrane is treated with soluble EFC large enzyme acceptor (EA) instead of an antibody, then EFC detection reagents are applied. At the locations where the protein band contains receptor-ED, a signal will be produced. The second assay is the addition of soluble EA and detection reagents to cells lysed in an assay microplate. A comparison between the parental cell line and the cell line expressing the orphan receptor will show the signal produced in response to EA, corresponding to the expression of the receptor. Data for both of these assay tests is found on the product datasheet.

Selecting Products

What is the difference between an eXpress kit and bioassay kit?

The ready-to-use eXpress Kits are used for research purposes only, and are implemented in early discovery phase for investigatory aims, screening applications, proof-of-concept, MOA confirmation, and rank-ordering studies. The eXpress kits are not recommended or supported for use in pre-clinical development phases including characterization or potency testing in QC lot release programs. Their protocols are optimized for convenience as well as precision and work well for high-throughput type workflows. They exclude a control ligand within the kit.
For biologics drug development programs, the ready-to-use Bioassay kit is the appropriate format implemented for characterization, detection of neutralizing antibodies, and potency testing in QC lot release applications. These bioassay kits have been developed, optimized, and qualified to meet market requirements or regulatory expectations. They are tightly controlled, engineered, and QC’d for maximum accuracy and precision as well as lot-to-lot and vial-to-vial consistency. Protocols can be more involved in order to seek this higher degree of stringency. The bioassay kits are packed with a control ligand, and some come pre-qualified with a marketed drug that targets their receptor.

For more information, refer to the Eurofins DiscoverX Assay Ready Kits webpage.

What detection kit should I use with my cell line?

Use the detection kit mentioned in the “Required Products” section of the product page or the cell line datasheet. The different detection kits contain different ingredients that are suited for a specific use, and not all assays are compatible with all detection kits, so swapping kits is not recommended. If you have a specific need or aim that you think may require changing detection kits, contact technical support for personalized help in selecting the right detection kit.

For more information, refer to the Eurofins DiscoverX Detection Kits and Reagents webpage.

Can HitHunter® cAMP assay kits be used with any cell line or only cAMP Hunter™ cell lines?

HitHunter cAMP assay kits can be used to measure cAMP production in any cell line that expresses GPCRs that signal via cAMP pathways. Some cell backgrounds (e.g. HEK 293) may have an inherently lower level of adenylyl cyclase and thus be poor producers of cAMP, but ultimately no tagging or genetic modification of the cells is required to use with this kit. cAMP Hunter cell lines are simply optimized to express good levels of adenylyl cyclase and the GPCR in question for best cAMP assay performance.

For more information, refer to the Eurofins DiscoverX cAMP Product Solutions webpage.

Can HitHunter cAMP assay kits be used with PathHunter® arrestin cell lines?

HitHunter cAMP assay kits use the same Enzyme Fragment Complementation (EFC) tags β-galactosidase enzyme donor (ED) and enzyme acceptor (EA)] as the PathHunter® arrestin assays (cell line plus detection reagents). As such, attempting to use HitHunter cAMP detection on PathHunter arrestin cell lines will not function properly, as the tags in each system will interfere with one another. If cAMP data on a PathHunter arrestin cell line is desired, non-EFC based third party cAMP kits should be used instead.

For more information, refer to the Eurofins DiscoverX cAMP Product Solutions and GPCR β-Arrestin Product Solutions webpages.

What type of readers can I use to perform a calcium assay on?

Calcium assays are best run on a FLIPR® series machine, or one of a few equivalents. The key characteristics for a calcium reader include: camera that can read all wells simultaneously on a less than 1 second interval (not a sequential scanning read like most plate readers) and an on-board reagent addition module with a full 96 (or 384) pin head. Calcium assays have very rapid dynamics, and sequential scanning or addition of agonist outside of the machine/one well at a time will result in missing the response and poor or no results.

Should I use clear or white bottom plates?

Clear bottom plates allow for examination of the cell health after plating and before assay, which is useful for troubleshooting a difficult assay. White bottom plates do not allow this, but also reduce possible spillover of signal between wells (usually only an issue in 384 or smaller formats, and when very bright and very dim wells are adjacent). For most uses, clear bottom plates are recommended due to the ability to see your cells.

For assays that use ligand presenting cell lines for induction, can soluble ligands be used instead?

Yes, if a soluble version of the ligand exists for a product that uses a ligand presenting cell line (such as a PathHunter Checkpoint Receptor cell lines), the soluble version should also activate the assay. The signal:background ratio, EC₅₀, and optimal conditions may differ, however.

Troubleshooting

Why are my cells replicating very slowly?

Engineered cells are known to proliferate more slowly than their unmodified parental cell would, so keep that in mind. Also, make sure you are using all the correct Eurofins DiscoverX media reagents for your cell line. If this is the case, other typical causes include:

Adding antibiotics to a thawed culture prematurely. This should be done only after the first passage has completed to allow cells to recover fully before selection pressure is applied.
Spinning down the thawed cells to remove DMSO. Eurofins DiscoverX thawing procedure uses dilution in large volumes of media instead, as this spin step unnecessarily stresses the cells.
Allowing cells to become over-confluent, causing contact inhibition of growth. This is often irreversible. Split cells at <90% confluence to avoid this.
Over-splitting. Some cell types, like U2OS, grow best with neighbors. If the culture is split very aggressively and becomes quite sparse, the cells might not grow as fast.
Clumping. Some cultures look less confluent than they are due to the cells’ tendency to clump together (HepG2 cells, for example). Trypsinizing cells and then replating all of them in a new tissue culture flask or doing a very small split ratio, can help cells spread so that they can grow faster or be counted more accurately.

What do I do if my EC50 or IC50 is right shifted?

If the right shift is not accompanied by a poor signal:background ratio, this typically indicates a problem with the ligand. Check your ligand source. If the ligand was bought from a third party vendor, it might be of poor quality or different potency or purity. Also, many ligands have a limited lifespan once diluted in solvent or if stored at 4°C. Typically this will be two weeks to a month. After this point, potency can decline. In addition, do not freeze-thaw ligands when avoidable. We recommend making single use aliquots to be kept frozen.
Make sure that your ligand dilutions were calculated properly, and that the ligand was fully in solution. If the ligand is somewhat precipitated, your “actual dose” in the well will not match what you thought you were adding.
For difficult ligands that do not go into solution in aqueous solvents, try adding DMSO. The Eurofins DiscoverX cellular assays can tolerate up to 2% DMSO in the final well volume if needed (although 0.5% or less is generally sufficient for most ligands).
If you are diluting your ligand in PBS, try adding 0.1% BSA. The additional protein can prevent the ligand from sticking to plastics, maintaining a more constant concentration.

What do I do if my signal:background is too low?

If the signal:background ratio is significantly lower than the reference for that ligand (more than 20-30% lower) check the following:

Check cell health. Make sure cells arrived frozen and that all procedures for culture or plating were followed. Cells should be attached in the assay plate’s wells and look healthy, with minimal dead cells. Some rounding may be normal for certain targets that are plated in low serum conditions – if in doubt, contact technical support.
Check your ligand by following the steps listed in ‘What do I do if my EC₅₀ or IC₅₀ is right shifted?’ question above.
If you are using crude lysates, the high protein content may be interfering with the assay. Try exchanging the well contents for fresh cell plating reagent just prior to the reading step.

Why is my relative light unit (RLU) value not equal to that on the reference curve?

RLU is literally relative, and every reader’s mathematical calculation of these values varies. Your reader likely defines “1 RLU” differently than Eurofins DiscoverX does. It is the signal-to-background ratio, and not the absolute RLU values, that you should aim to match.

Relatedly, RLU from the same reader may vary day-to-day based on environmental conditions and uptime of the reader.
If your RLU values are wildly different from what is usual for your reader, such as being in the hundreds of millions, check for bacterial contamination. Bacteria that contain the β-galactosidase enzyme that is used in Enzyme Fragment Complementation (EFC) assay can produce strong false signals.

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