IL-12 Dual Reporter HEK 293 Cells (SEAP & GFP)

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IL-12 responsive STAT4-SEAP & STAT4-GFP reporter assay

Signaling in HEK-Blue™ GFP-IL-12 cells

HEK-Blue-GFP™ IL-12 cells are designed to monitor human IL-12-induced STAT4 stimulation or inhibition using GFP and SEAP reporters. This fluorescent and colorimetric bioassay can be used for screening activatory molecules, such as engineered cytokines, or inhibitory molecules, such as neutralizing antibodies.
The STAT4-activation dual readout allows:
- Real-time monitoring by assessing GFP fluorescence with standard plate readers or live-cell imaging systems.
- Quantification by measuring SEAP activity in the culture supernatant using the QUANTI-Blue™ Solution.

HEK-Blue-GFP™ IL-12 cells respond specifically to recombinant human (h)IL-12 and mouse (m)IL-12. Their reliable and consistent performance makes them suitable for release assays of therapeutic molecules that inhibit IL-12 signaling, such as Ustekinumab, a monoclonal antibody targeting the IL-12 p40 subunit.  Importantly, both reporters maintain equivalent sensitivity, ensuring no loss in assay performance (see figures).

Key features

• Fully functional IL-12 signaling pathway
• HTS ready: STAT4-inducible GFP fluorescence, no substrate addition
• Quantitative STAT4-inducible SEAP reporter readout
• High sensitivity to human (h) and mouse (m) IL-12 activity
• Stability guaranteed for 20 passages

Applications

• Therapeutic development
• Drug screening
• Release assay

Interleukin 12 (IL-12) is a pleiotropic cytokine driving IFN-γ production, proliferation of activated T and NK cells, and differentiation of Th1 cells in response to microbial infections. Dysregulated IL-12 production is linked to immune-mediated inflammatory diseases such as psoriasis, rheumatoid arthritis, Crohn's disease, and multiple sclerosis. 

 More details

InvivoGen’s products are for internal research use only and not for clinical or veterinary use.

Figures

Dose-response of HEK-Blue-GFP™ IL-12 cells to recombinant IL-12. Cells were incubated overnight with increasing concentrations of recombinant human IL-12 (hIL-12) and murine IL-12 (mIL-12). (A) GFP mean fluoresence intensity was measured directly in the culture wells using a fluorescence plate reader. Data are shown as mean ±  SEM). (B) SEAP activity was measured in the culture supernatant using QUANTI‑Blue™ Solution, a SEAP detection reagent. The optical density (OD) at 650 nm is shown as mean ± SEM.

HEK-Blue™ IL-12 parental cells (left panels) and HEK-Blue-GFP ™ IL-12 cells (right panels) were incubated overnight with increasing concentrationsof recombinant human IL-12. The GFP mean fluorescence intensity was assessed using flow cytometry.

HEK-Blue™ IL-12 parental cells (left panels) and HEK-Blue-GFP ™ IL-12 cells (right panels) were incubated overnight with 3 ng/ml of recombinant human IL-12. The GFP expression was assessed using fluorescence microscopy. The top panels show the cells with white light. The bottom panels show the cells with the GFP filter. Scale bars: 100 µm.

Dose-dependent inhibition of HEK-Blue-GFP™ IL-12 cell response using Ustekinumab biosimilar. Increasing concentrations of Anti-hIL-12/IL-23-p40-hIgG1 (0.1 ng/ml - 10 µg/ml) were incubated with recombinant human IL-12 (A: 3 ng/ml, B: 1 ng/ml) for 1 h before the addition of HEK-Blue-GFP™ IL-12 cells. After overnight incubation, (A) GFP mean fluoresence intensity was measured directly in the culture wells using a fluorescence plate reader and (B) SEAP activity in the cell culture supernatant was assessed using QUANTI-Blue™ Solution. Data are shown in percentage of activity (mean ± SEM).

Response of HEK-Blue-GFP™ IL-12 cells to a panel of cytokines. Cells were stimulated with various human (h) and mouse (m) recombinant cytokines: 10 ng/ml of hIL-12 or mIL-12, and 100 ng/ml of hIL-23, mIL-23, hIL-27, hIL-35, hTNF-α, hIFN-α, and hIFN-γ. After overnight incubation, (A) GFP mean fluorescence intensity was measured directly in the culture wells using a fluorescence plate reader, and (B) SEAP activity in the cell culture supernatant was assessed using QUANTI-Blue™ Solution. Data are shown in percentage of activity (mean ± SEM).

Specifications

Cell type: Epithelial

Tissue origin: Human Embryonic Kidney

Target: IL-12

Specificity: Human, Mouse

Reporter genes: SEAP, GFP

Antibiotic resistance: Blasticidin, Zeocin®, Hygromycin, Puromycin

Detection range: 1 ng/ml - 100 ng/ml (hIL-12 and mIL-12) — Note: both GFP and SEAP reporters maintain equivalent sensitivity, ensuring no loss in assay performance

Growth medium: Complete DMEM (see TDS)

Growth properties: Adherent

Mycoplasma-free: Verified using Plasmotest™

Quality control: Each lot is functionally tested and validated.

Contents

HEK-Blue-GFP™ IL-12 Cells (hkbg-il12)

• 1 vial containing 3-7 x 10⁶ cells
• 2 x 1 ml HEK-Blue™ Selection (250x concentrate)
• 1 ml of Puromycin (10 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)
   

HEK-Blue-GFP™ IL-12 vial (hkbg-il12-av)

• 1 vial containing 3-7 x 10⁶ cells

Shipped on dry ice (Europe, USA, Canada, and some areas in Asia)

Notification:  Reference #hkbg-il12-av can only be ordered together with reference #hkbg-il12.

Details

Cell line description

HEK-Blue-GFP™ IL-12 cells are derived from the HEK-Blue™ IL-12 cell line featuring the human IL-12 receptor and a STAT4-SEAP reporter. They were further engineered by the addition of a STAT4-GFP reporter. 

The binding of IL-12 to its receptor triggers a signaling cascade leading to STAT4 activation and the subsequent production of GFP and SEAP. Importantly, both reporters maintain equivalent sensitivity, ensuring no loss in assay performance.

•  The GFP fluorescence can be assessed using standard plate readers or live-cell imaging systems.
•  The SEAP activity in the culture supernatant can be measured using QUANTI-Blue™ Solution, a SEAP detection reagent.

HEK-Blue-GFP™  IL-12 cells detect human (h) IL-12 and mouse (m) IL-12. These cells also respond, to a weaker extent, to hIL-27 and hIFN-α. However, they do not respond to other STAT4-signaling cytokines of the IL-12 cytokine receptor family: IL-23 and IL-35 (see figures).

IL-12 background

Interleukin 12 (IL-12), also known as IL-12p70, cytotoxic lymphocyte maturation factor (CLMF), or natural killer cell stimulatory factor (NKSF) is the founding member of the heterodimeric IL-12 cytokine family. This family is characterized by the sharing of p19, p28, p35, p40, and Ebi3 subunits, and includes IL-12, IL-23, IL-27, IL-35, and IL-39 [1, 2]. The secretion of bioactive IL-12 requires the co-expression of IL-12p35 and IL-12p40 and disulfide bond formation. IL-12 is primarily produced by pathogen-activated macrophages and dendritic cells (DC) [1, 2].

The binding of IL-12 to its IL-12Rβ1/IL-12Rβ2 heterodimeric receptor triggers a JAK2/TYK2 signal transduction leading to the activation of STAT4. Activated STAT4 forms homodimers that are translocated to the nucleus where they regulate the expression of target genes. Subsequent gene expression promotes the cytotoxic activity of Natural Killer (NK) and CD8+ T cells, and drives IFN-γ production by NK and activated CD4+ T cells [1, 2]. IFN-γ contributes to the differentiation of Th1 cells and inhibits Th2 cell development. Moreover, it enhances DC maturation and antigen presentation.

Interestingly, secreted IL-12p40 monomers and homodimers (IL-12p80) have also been found and described to function as IL-12 antagonists or agonists, depending on the experimental studies [2].

Relevance for therapeutics development

IL-12 is a key cytokine for controlling intracellular infections. However, excessive IL-12 production contributes to several immune-mediated inflammation diseases including rheumatoid arthritis, type I diabetes, multiple sclerosis, and Crohn's disease [1].

Ustekinumab is a fully human monoclonal antibody (mAb) that targets the p40 subunit common to IL-12 and IL-23, preventing their interactions with their receptor common chain IL-12Rβ1 [3]. This antibody is FDA-approved for treating psoriasis, psoriatic arthritis, Crohn's disease, and ulcerative colitis [4]. Addressing the benefits of neutralizing IL-12 and IL-23 together or individually, depending on the clinical context can be achieved using neutralizing mAbs targeting the p19 subunit of IL-23 [5].

As IL-12 drives strong pro-inflammatory and cytotoxic activities, a series of IL-12-based products have been explored for cancer therapy. These strategies must control targeted delivery to achieve high local IL-12 while avoiding systemic toxicity [6-8].

References:

1. Dembic., Z., 2015. Chapter 6 - Cytokines of the immune system: Interleukins. The cytokines of the immune system (book): 143-239.
2. Floss, D.M., et al., 2020. IL-12 and IL-23-Close Relatives with Structural Homologies but Distinct Immunological Functions. Cells, 9(10): 2184.
3. Benson, J.M, et al., 2011. Discovery and mechanism of ustekinumab: a human monoclonal antibody targeting interleukin-12 and interleukin-23 for treatment of immune-mediated disorders. MAbs 3(6):535-545.
4. FDA website accessed in Nov 2024: https://www.accessdata.fda.gov/drugsatfda_docs/label/2024/761044s013lbl.pdf#page=37
5. Tait Wojno, E.D. et al., 2019. The Immunobiology of the Interleukin-12 Family: Room for Discovery. Immunity. 50(4):851-870.
6. Leonard, W.J. & Lin, J. X., 2023. Strategies to therapeutically modulate cytokine action. Nat Rev Drug Discov. 22(10):827-584.
7. Briukhovetska D., et al., 2021. Interleukins in cancer: from biology to therapy. Nat Rev Cancer. 21(8):481-499.
8. Yi, M., et al., 2024. Targeting cytokine and chemokine signaling pâthways for cancer therapy. Signal Transduction and Targeted Therapy. 9(1):176.

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