Human STING-KO Reporter HEK 293 Cells

In general, InvivoGen’s cells are shipped on dry-ice and since dry-ice is not nearly as cold as liquid nitrogen, thawing of the cells technically begins during transport. Thus, we recommend a full thaw upon receipt to ensure the best recovery results.

NOTE: this is not applicable in some countries in Asia where cell lines are sent at room temperature using our specially designed flask

The most important step is the thawing procedure upon receipt of the cells. 

Do not put your cells at -80°C or in liquid nitrogen as this may damage the cells. 

You must propagate the cells immediately upon receipt.

Below are a few tips we recommend if you encounter any issues during initial culture:
• 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 the 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.

If you are working with THP1 cells in particular, here are a few additional tips:
• THP1 cells need to be passaged between 3 x 10⁵ and 7 x 10⁵ cells per mL (we recommend 5 x 10⁵ cells per ml). Below this, your cells will take a long time to grow and above 2 x 10⁶ cells per ml you may have toxicity.
• Between each passage, do not centrifuge the cells. THP1 cells grow better in conditioned media, therefore when passaging cells, leave 1 – 2 ml of the old media and add fresh media on top. Do not leave more than 50% of conditioned media as the cells will not have enough nutrients to properly grow.

After thawing, the cells can be more sensitive to selective antibiotics due to the initial low levels of resistance markers. Therefore, it is recommended to wait for 2 - 3 passages before adding the selective antibiotics in order to avoid further stress to the cells during the first couple of passages.

Both Glutamine and GlutaMax have been tested in-house and can be used, with no difference in the cells noted.

Many of InvivoGen’s cell lines are engineered with a SEAP (Secreted Embryonic Alkaline Phosphatase) reporter system, and FBS, in many cases, contains traces of alkaline phosphatase (AP) that interferes with the test results. Therefore, to avoid background noise we recommend working with inactivated serum with all of our cells.

In house we inactivate serum by heating at 56°C for 30 minutes. It is important to wait for the water bath to reach the 56°C before placing the tube of serum in to ensure the serum is fully inactivated.

In house we always use heat-inactivated serum for the freezing medium, growth medium and test medium. However, please note that it is not a problem if you wish to use non-heat-inactivated in the freezing medium.

It is important to choose an endotoxin-free serum for the culture of our reporter cell lines. InvivoGen systematically asks for the Certificate of Analysis of different batches of serum to ensure a low level of endotoxin as to not activate the NFkB pathway.

100 U/ml Penicillin and 100 µg/ml Streptomycin

No, this shouldn’t be a problem as long as there is at least 2 mM L-Glutamine in the medium. Most DMEM formulations already contain 4mM of L-Glutamine. However, if you need to add L-Glutamine to your medium you will only need to add 2 mM for the cells to grow properly.

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 medium and seed 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. Therefore, 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)

It isn’t normal for your cells to divide only once a week. Depending on the clone, they usually have a doubling time of 24 – 72 hours. THP1 cells must be cultured at quite a high concentration (at least 5 x 10⁵ cells/ml). Please note that our R&D teams have noticed that THP1 cells often die when they are diluted too harshly. Also, it may help to not add Normocin™ while waiting for improvement to their growth.

It is not a problem if the cells are clumping, as long as they are growing fine and have normal morphology. You may want to centrifuge the cells to get rid of the dead cells as sometimes this is the reason for clumping. Please note that we recommend homogenizing the cells before performing your assays.

We recommend to centrifuge at 120 G for 10 minutes.

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.

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.

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

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

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.

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.

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.

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 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).

It is hard to estimate the deviation factor regarding cell passage number. However, we note very little difference in our experimental results, with no more than the slight variations you expect due to handling errors.

Yes, InvivoGen’s reporter cell lines can be used in ELISA and Western Blots. However, please note that we do not sell them for this purpose.

They are semi-quantitative tests. However, they can be used for quantitative measurements by making a standard curve using a positive control.

If you cannot run the detection assays immediately, you can store supernatant samples for up to a week at 4°C. Supernatant samples are active for a longer time when kept at -20°C. However, in this case we would recommend supplementing the test medium with 20% FBS to protect the SEAP as much as possible during the freezing process. Do not repeat freeze/thaw cycles.

It depends on the inhibitor. If the inhibitor blocks a receptor or an element in the signaling pathway, we recommend pre-incubating the inhibitor with the cells for at least 30 minutes. If the inhibitor is binding or blocking the ligand (i.e. an antibody against a specific cytokine) then it is best to pre-incubate the inhibitor with the ligand prior to adding to the cells.

In house the cells are seeded at the same time as adding the ligand (ligand first).

To limit the activation of NFκB before stimulation (lower background):
• Use healthy cells that have been passaged at least 24 hours before the assay
• Do not allow cells to be greater than 80 % confluent
• Use pre-warmed PBS to rinse your cells
• Use heat-inactivated FBS (to eliminate residual alkaline phosphatase in the serum)
• Do not centrifuge the cells prior to stimulation
• Rather than trypsin, use PBS for 2 - 3 min @ 37°C and gently pipet up and down to detach cells 
• Do not use an excessive number of cells per well
(approximately 50 000 cells/well as recommended on the TDS).

We recommend to not use Normocin™ in the test medium with all of our cell lines as it is better to reduce the number of potential interfering agents in the medium when performing the assay. The same goes for the use of selective antibiotics

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.

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

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

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.

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

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.

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.

For adherent cells, we recommend DMEM, 20% (v/v) fetal bovine serum (FBS), and 10% (v/v) DMSO.

For suspension cells, we recommend fetal bovine serum (FBS) and 5-10% (v/v) DMSO

We highly recommend keeping a flask of cells running until you begin thawing your frozen stock in case something has gone wrong during the freezing step.

Our reporter cells require the activation of just one transcription factor (NFκB and/or AP-1) to produce a signal.

InvivoGen’s Lucia® luciferase is completely synthetic and is not related to any natural luciferase.

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

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).

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

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

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

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

The difference in activity is approximately 10-fold.

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

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.

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.

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

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.

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.