IFN-γ Reporter HEK 293 Cells

STAT1-SEAP reporter cells

ABOUT

IFN-γ responsive STAT1-SEAP reporter cells

HEK-Blue™ IFN-γ cells are designed to monitor human type II interferon IFN-γ-induced STAT1 stimulation or inhibition. This colorimetric bioassay can be used for screening activatory molecules, such as engineered cytokines, or inhibitory molecules, such as neutralizing antibodies.

HEK-Blue™ IFN-γ cells respond specifically to recombinant human IFN-γ. The reliable and consistent performance of HEK-Blue™ IFN-γ cells makes them suitable for release assays of therapeutic molecules that inhibit IFN-γ signaling, such as Fontolizumab, a monoclonal antibody that targets IFN-γ and prevents its binding to its receptor (see figures).

Key Features

  • Readily assessable STAT1-SEAP reporter activity
  • Convenient readout using  QUANTI-Blue™ Solution
  • High sensitivity to human (h) IFN-γ activity
  • Stability guaranteed for 20 passages

Applications

  • Therapeutic development
  • Drug screening
  • Release assay

 

Interferon-gamma (IFN-γ) is a pro-inflammatory cytokine that plays a key role in innate and adaptive immune responses to intracellular pathogens and tumor immunosurveillance.

More details

Disclaimer:  These cells are for internal research use only and are covered by a Limited Use License (See Terms and Conditions). Additional rights may be available.

Free cytokine
Signaling pathway in HEK-Blue™ IFN-γ cells
Signaling pathway in HEK-Blue™ IFN-γ cells

SPECIFICATIONS

Specifications

Target

IFN-γ

Target species

Human

Tested applications

Detection and quantification of human IFN-γ

Cell type
Epithelial
Growth properties
Adherent
Tissue origin
Human embryonic kidney cells
Reporter gene
SEAP
Detection method
Colorimetric
Detection range

0.1 ng/ml -10 ng/ml

Antibiotic resistance
Blasticidin
Zeocin®
Growth medium

Complete DMEM (see TDS)

Mycoplasma-free

Verified using Plasmotest™

Quality control

Each lot is functionally tested and validated.

CONTENTS

Contents

  • Product: 
    HEK-Blue™ IFN-γ Cells
  • Cat code: 
    hkb-ifng
  • Quantity: 
    3-7 x 10^6 cells
Includes:
  • 1 ml of Blasticidin (10 mg/ml)
  • 1 ml of Zeocin® (100 mg/ml)
  • 1 ml Normocin® (50 mg/ml)
  • 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent)

Shipping & Storage

  • Shipping method:  Dry ice

    • Storage:

    • Liquid nitrogen vapor
    Stability: 20 passages

    Caution:

    • Upon receipt, store immediately in liquid nitrogen vapor. Do not store cell vials at -80°C.

Details

Cell line description

HEK-Blue™ IFN-γ cells were generated by stable transfection of the human embryonic kidney HEK293 cell line with the gene encoding the human STAT1 to obtain a fully active type II IFN signaling pathway. The other genes of the pathway (IFNGR1, IFNGR2, JAK1, and JAK2) are naturally expressed by these cells. In addition, a STAT1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene was introduced. The binding of IFN-γ to its receptor triggers a signaling cascade leading to STAT1 activation and the subsequent production of SEAP. This can be readily assessed in the supernatant using QUANTI-Blue™ Solution, a SEAP detection reagent.

HEK-Blue™ IFN-γ cells detect human (h) IFN-γ, but not mouse (m) IFN-γ. Of note, these cells do not respond to either type I IFNs (IFN-α/β) or type III IFNs (IFN-λ) (see figures).
 

IFN-γ background

IFN-γ, also known as Type II IFN or immune interferon, is predominantly produced by innate immune cells, such as Natural Killer (NK) cells and innate lymphoid type 1 cells (ILC1), and activated adaptive immune cells, such as Th1 CD4+ T cells and cytotoxic CD8+ T cells [1]. This cytokine is produced as a secreted homodimeric molecule in response to infections and growing tumors [1, 2]. IFN-γ engages a receptor composed of two IFN-γR1 chains and two IFNγ-R2a, thus forming a hexameric complex [2]. While IFN-γR1 is constitutively expressed on all nucleated cells, the expression of IFNγ-R2 is tightly regulated. The binding of IFN-γ to its receptors triggers a JAK1/JAK2 signal transduction leading to the activation of STAT1. Activated STAT1 forms homodimers that are translocated to the nucleus where they bind interferon-gamma-activated sites (GAS) in the promoter of interferon-stimulated genes (ISGs). ISGs encode many products with direct effector or regulatory immune functions [1]. Thus IFN-γ plays a versatile role in immune responses and tissue homeostasis [1].

 

Relevance for therapeutics development

IFN-γ contributes to the pathogenesis of inflammatory diseases, such as Crohn's disease, psoriasis, haemophagocytic lymphohistiocytosis (HLH), and macrophage activation syndrome (MAS) [3-5].
Fontolizumab (aka HuZAF™) is a therapeutic, humanized monoclonal antibody (mAb) that targets IFN-γ. By binding to IFN-γ, Fontolizumab prevents it from interacting with its receptor (IFN-γR) on the surface of immune cells, thus inhibiting downstream inflammatory response. Fontolizumab was used as an immunosuppressive drug to treat inflammatory diseases, including Crohn's disease and psoriasis. While the mode of action was promising, clinical trials did not demonstrate sufficient clinical benefit [4, 5].
Emapalumab is a fully human monoclonal antibody (mAb) that targets both free and receptor-bound IFN-γ, preventing downstream signaling. This therapeutic antibody was FDA-approved in 2018 for treating pediatric and adult patients with HLH [3]. 

The administration of recombinant IFN-γ is a strategy for enhancing anti-infectious and anti-tumoral innate and adaptive immune responses.
Interferon gamma-1b (Actimmune®) is a form of recombinant human IFN-γ approved by the FDA to treat infections associated with chronic granulomatous disease and to slow the progression of severe malignant osteopetrosis [6]. IFN-γ monotherapy to treat cancer has been of limited success. This is partly explained by IFN-γ's short half-life and dual anti- and pro-tumor activities [7, 8]. Multiple clinical trials are ongoing to explore the combination of IFN-γ with other cancer therapeutics [8].
Another strategy relies on the engineering of IFN-γ partial agonists to tune IFN-γ receptor signaling output. Interestingly, such recombinant IFN-γ variants can exhibit biased gene-expression profiles, such as the retention of upregulation of class I molecules and impaired induction of inhibitory checkpoint molecules by cancer cells [2]. These results demonstrate that the two opposing functions of IFN-γ in the tumor microenvironment can be decoupled, offering a route for therapeutic applications.

 

References:

1. Ivashkiv L.B., 2018. IFNγ: signalling, epigenetics and roles in immunity, metabolism, disease and cancer immunotherapy. Nat Rev Immunol. 18(9):545-558
2. Mendoza, J.L., et al., 2019. Structure of the IFNγ receptor complex guides design of biased agonists. Nature. 567(7746):56-60.
3. Vallurupalli M. & Berliner N., 2019. Emapalumab for the treatment of relapsed/refractory hemophagocytic lymphohistiocytosis. Blood. 134(21):1783-1786.
4. Reinisch W et al., 2009. Fontolizumab in moderate to severe Crohn's disease: A phase 2, randomized, double-blind, placebo-controlled, multiple-dose study. Infl Bowel Dis., 16(2):233-242.
5. Harden J.L., et al., 2015. Humanized anti-IFNg (HuZAF) in the treatment of psoriasis. J. Aller Clin Immunol. 135(2):553.
6. Silva, A.C. & Lobo, J.M. Sousa., 2020. Cytokines and growth factors. Current Applications of Pharmaceutical Biotechnology. 87-113.
7. Castro, F., et al., 2018. Interferon-Gamma at the Crossroads of Tumor Immune Surveillance or Evasion. Front Immunol. 9:847.
8. Yi, M., et al., 2024. Targeting cytokine and chemokine signaling pathways for cancer therapy. Signal Transduction and Targeted Therapy. 9(1):176.

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CUSTOMER SERVICE & TECHNICAL SUPPORT

Question about this product ?

FAQ

HEK293 cell line description

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.

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.

Cell line culture

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.

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).

Assays

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.

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.