HEK293 cell lines

Cell line description

Where are endosomal TLRs (TLR3, TLR7, TLR8 and TLR9) expressed in the HEK-Blue™ cells?

We presume them to be in the endosome as we express the wild-type, full-length genes. Additionally, their signal can be blocked by Chloroquine, an endosomal acidification inhibitor. However, as these TLRs are over-expressed there may potentially be low expression of them on the cell surface.

Do HEK293 cells express any endogenous Pattern Recognition Receptors (PRRs)?

HEK293 cells express TLR1, TLR3, TLR5, TLR6, and NOD1.
They respond to TLR3, TLR5, and NOD1 agonists, but at a much lower level compared to HEK293 cells transfected with these receptors.

Do you have any information about the GM classification of HEK293 transfected cell lines?

In the United States, HEK293 cell lines are designated Biosafety Level 2 according to the Center for Disease Control and Prevention (CDC).
In Germany, HEK293 cell lines are designated Biosafety Level 1 according to the Central Committee of Biological Safety, Zentrale Kommission für die Biologische Sicherheit (ZKBS).
You can check with your country’s regulatory authority regarding the use of these cells.
Please note that there is no replicating/infectious Adenovirus 5 in these cells.

What is the difference between the HEK-Blue™ Null1 and HEK-Blue™ Null2 cells (parental cell lines of the HEK-Blue™ cells)?

The minimal promoter isn't the same but the difference in expression for these two Null cell lines is minor.

Regarding the construction of InvivoGen’s reporter cell lines, how are the plasmids stably integrated into the HEK-Blue™ cells?

There is no specific integration system used to generate our stable cell lines.
The selection pressure is enough to obtain stable clones. The receptors are added by simple transfection of plasmids using a cationic lipidic transfection agent (the plasmids are not linearized before transfection).

Do you have a variety of HEK-Blue™ Null cells that are not resistant to G418?

Only our HEK-Blue™ Null1-k and Null2-k cells are sensitive to G418.

Do you have a variety of HEK-Blue™ Null cells that are not resistant to Puromycin?

The only HEK-Blue™ Null cells that are sensitive to Puromycin are the HEK-Blue™ Null1-v cells.

Do you have HEK-Blue™ Null cells that are not resistant to Puromycin?

The only HEK-Blue™ Null cells that are sensitive to Puromycin are the HEK-Blue™ Null1-v cells.

Do the HEK-Blue™ cells express Fc receptors?

Our RNAseq data confirms that our HEK-Blue™ cells express FcRN, however, this has not been functionally tested.

What is the difference in activity between an overexpressed TLR and an endogenous TLR in the HEK-Blue™ cells?

The difference in activity is approximately 10-fold

What is the difference between the 293-TLR cells and HEK-Blue™ TLR cells?

Both cell types overexpress a designated TLR. The primary difference is that HEK-Blue™ cells include the NFκB inducible SEAP reporter construct.

What is the difference between HEK-Blue™ cells and HEK-Dual™ cells?

HEK-Blue™ cells only express a single NFκB inducible SEAP reporter gene. Whereas, HEK-Dual™ cells have the addition of the Lucia™ gene knocked into the IL-8 locus. Thus, when IL-8 is activated following stimulation, HEK-Dual™ cells can report this with the secretion of Lucia™ luciferase.
It should also be noted that the HEK-Dual™ cells have been knocked out for TLR3, TLR5, and TNFR to limit interference from other TLRs when studying a specific TLR pathway.

What is the difference between the HEK-Blue™ IL-1R and HEK-Blue™ IL-1β cells?

HEK-Blue™ IL-1R cells express both human and murine IL-1β receptors, thus can detect both species.
On the other hand, HEK-Blue™ IL-1β cells are specific for human IL-1β, but can still detect higher concentrations of mouse IL-1β.

Cell line culture

What is the split ratio you recommend for the HEK Blue™ cells once the vial has grown confluent?

The split ratio will depend on when you expect confluency. Typically, the doubling time of HEK-Blue™ cells is approximately 24 hours.
Therefore, if you use a split ratio of 1:2 (50%) into a new flask, cells should be confluent the following day. If you use a split ratio of 1:4 (25%) you can expect the cells to be confluent after 2 days.

How much would you advise to use from the HEK-Blue™ Selection antibiotic?

Our HEK-Blue™ Selection is provided in 1ml tubes with each containing a 250X solution.
Therefore, you should dilute HEK-Blue™ Selection 1:250 into your media to have a 1X concentration.

At what density should HEK-Blue™ cells be plated?

HEK-Blue™ cells should be seeded at a density of approximately 1.5 x 106 cells/ml in a T25 flask or 4 – 5 x 106 cells/ml in a T75 flask.

What cell culture plate do you recommend to use with the HEK-Blue™ reporter cells?

We recommend using a flat-bottom, clear walled cell culture plate.

I’m having trouble growing HEK cells, do you have any tips for my initial culture?

Below are a few tips we recommend to help get your HEK cells growing:
• For the first 2-3 passages, grow cells in media containing 20% FBS and no antibiotics.
• Do not allow cells to reach 100% confluency Please check cells as regularly as possible.
• The cells should not be grown in 20% FBS for too long. Use media with 10% FBS after 2 or 3 passages.
• When making frozen stocks, continue growing additional cultures in case there is a problem with the frozen stock.

Why is Trypsin not recommended for detaching HEK-Blue™ cells?

Trypsin does not adversely affect the health or growth of these cells. However, it is known that high concentrations will occasionally induce the activation of NFκB resulting in a higher background in your assay.
Moreover, we have observed some cases where trypsin has been contaminated with TLR2, TLR4, and TLR5 contaminants, which can also interfere with the assay results.

I have a few different HEK-Blue™ TLR cells. Why are they growing at different rates?

It is not unusual for different TLR cells to grow at different rates. Some TLR clones happen to grow a little slower/faster than others. This is often clone dependent.

My HEK-Blue™ cells are not properly attaching to the bottom of the flask. What type of plastic equipment do you recommend?

When the HEK-Blue™ cells are non-adherent, either they were diluted too harshly at the start or they have grown over-confluent in a small flask and suffocated.
To avoid this in the future:
• Change the media and plate the cells at a density of approximately 1.5 x 106 cells/ml in a T25 flask.
• Wash the cells before putting them into a new flask. Sometimes when the cells are non-adherent, it is due to the clustering of both live and dead cells. Additionally, this will get rid of any remaining DMSO which could affect the adhesion of the cells to the flask.
• Use medium with 20% FBS.
• The use of CellBIND flasks can sometimes help to increase attachment and growth of the cells (however CellBIND flasks are not required in the normal protocol).


After performing the assay, all wells in the plate are blue, even the negative control. What could be the problem?

There are 2 possible explanations as to why a blue color is observed in all wells.

1. It could be due to the presence of Alkaline Phosphatase (AP) in the culture medium. To see if this is the case, there is a very simple test to perform. Add 50 µl of the medium used for cell culture (without cells) and 200 µl of resuspended HEK-Blue™ detection medium or QUANTI-Blue™. If the medium turns blue, then it is due to the presence of Alkaline Phosphatase (AP) in the serum of the media. In this case, you must heat the serum to inactivate the AP and repeat the medium test. At this point the test should give a negative result (no blue color).

2. It could be due to improper handling of cells before the test. To avoid activation of NFκB before stimulation and reduce the risk of false positive results:
• Use pre-warmed PBS to wash cells
• Use heat-inactivated FBS
• Do not centrifuge cells prior to stimulation
• Do not use trypsin

Can we use HEK-Blue™ Detection instead of QUANTI-Blue™ for the HEK-Blue™ cytokine reporter cells?

We have noticed a loss of sensitivity when using HEK-Blue™ Detection medium instead of QUANTI-Blue™ on our cytokine reporter cell lines.
Therefore, we recommend using QUANTI-Blue™, which is provided with the cells, as this is what we use in house.

Can we use HEK-Blue™ Selection during assays?

We recommend to not use any antibiotics at all during assays to ensure the least amount of potential interfering agents in the medium.
Therefore, we do not add HEK-Blue™ Selection to the test media.

Can the HEK-Blue™ reporter cell lines be used for plasma or serum samples? If so, what provides the best results, plasma or serum samples?

We have only tested the use of plasma and serum samples on our HEK-Blue™ hTLR2 cell line.
The results demonstrated that when compared to using standard samples (in DMEM), serum samples give a single log difference.
On the other hand, we found a 3-log difference between DMEM and plasma samples.
This is why we would recommend using serum samples over plasma samples.

Can HEK-Blue™ Detection and QUANTI-Blue™ both be used on HEK-Blue™ TLR cells?

Yes, they can be used interchangeably. However, please note that the protocols are distinctly different and need to be followed accordingly.

How easy are HEK293 cells to transfect?

HEK293 cells are very easy to transfect with a transfection efficiency of approximately 80%.

Is it possible to see HEK-Blue™ Detection assay results in less than 16 hours?

It depends on the cell line and the concentration of the ligand used to stimulate the cells. In general, we record the results following 16 – 24 hours of stimulation.

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