CD40L Reporter HEK 293 Cells

Notification:  Reference #hkb-cd40-av can only be ordered together with reference #hkb-cd40.

CD40L responsive NF-κB-SEAP reporter assay

Signaling pathway in HEK-Blue™ CD40L cells

HEK-Blue™ CD40L cells are designed to monitor human CD40L-induced NF-κB stimulation or inhibition. This colorimetric bioassay can be used to screen activatory or inhibitory molecules, such as engineered cytokines and neutralizing antibodies, respectively.

HEK-Blue™ CD40L cells respond specifically to recombinant human CD40L and mouse CD40L. Their reliable and consistent performance makes them suitable for release assays of activatory and inhibitory molecules such as Frexalimab, a monoclonal antibody that targets CD40L and prevents its binding to its receptor (see figures).

Key features

• Readily assessable NF-κB reporter activity
• Convenient readout using QUANTI-Blue™ Solution
• High sensitivity to human (h)CD40L and mouse (m)CD40L
• Stability guaranteed for 20 passages

Applications

• Therapeutic development
• Drug screening
• Release assay

CD40L is a protein that is primarily expressed on activated T cells and is a member of the tumor necrosis factor superfamily. Of note, CD40L, together with its receptor CD40, plays a pivotal role in cellular and humoral immunity.

 More details

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

Figures

Dose-response of HEK-Blue™ CD40L cells to recombinant CD40L. Cells were stimulated with increasing concentrations of recombinant human (h)CD40L. After overnight incubation, the response was determined using QUANTI-Blue™, a SEAP detection reagent. The optical density (OD) at 650 nm is shown as mean ± SEM.

Response of HEK-Blue™ CD40L cells to a panel of cytokines. Cells were stimulated with various human recombinant cytokines: hIFN-α2b or hIFN-β-1a (1000 U/ml), hIFN-γ (1 µg/ml), hIL-1β (1 ng/ml), hIL-4 (100 ng/ml), hIL-6 (100 ng/ml), hIL-13 (100 ng/ml), hIL-18 (100 ng/ml), hTNF-α (1 ng/ml), hTGF-b1 (100 ng/ml), hCD40L (100 ng/ml), and mCD40L (100 ng/ml). After overnight incubation, SEAP activity was assessed using QUANTI-Blue™. The OD at 650 nm is shown as mean ± SEM.

Dose-dependent inhibition of HEK-Blue™ CD40L cell response using Anti-hCD40L-IgG mAb. A serial dilution of Anti-hCD40L-hIgG monoclonal antibody (mAb) was incubated with 100 ng/ml of recombinant human CD40L for 30 minutes prior to the addition of the HEK-Blue™ CD40L cells. After overnight incubation, the SEAP response was determined using QUANTI-Blue™, a SEAP detection reagent. Data are presented as percentage of neutralization (mean ± SEM).

Specifications

Cell type: Epithelial

Tissue origin: Human Embryonic Kidney

Target: CD40L

Specificity: Human, Mouse

Reporter gene: SEAP

Antibiotic resistance: Blasticidin, Zeocin®

Detection range: 3 ng/ml - 1 µg/ml (hCD40L and mCD40L)

Growth medium: Complete DMEM (see TDS)

Growth properties: Adherent

Mycoplasma-free: Verified using Plasmotest™

Quality control: Each lot is functionally tested and validated.

Contents

• 1 vial containing 3-7 x 10⁶ cells
• 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)

Shipped on dry ice (Europe, USA, Canada)

Details

CD40 Ligand (CD40L), also known as CD154, TRAP, or gp39, is a type II transmembrane glycoprotein belonging to the tumor necrosis factor (TNF) family. It is mainly expressed in CD4+-T cells and interacts with CD40 on antigen-presenting cells to regulate both humoral and cellular immune responses [1-3].

The CD40 cytoplasmic domain binds directly to several TNF receptor-associated factors (TRAFs), and this interaction is thought to initiate CD40 signaling. CD40-mediated signaling results in NF-κB, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) activation. CD40L-CD40 interactions are thought to play an important role in the pathogenesis of many diseases [4, 5].

1. Karnell J.L. et al., 2019. Targeting the CD40-CD40L pathway in autoimmune diseases: Humoral immunity and beyond. Adv Drug Deliv Rev. 141:92-103.
2. Laman J.D. et al., 2017. Functions of CD40 and Its Ligand, gp39 (CD40L). Crit Rev Immunol. 37(2-6):371-420.
3. Elgueta R. et al., 2009. Molecular mechanism and function of CD40/CD40L engagement in the immune system. Immunol. Rev. 229, 152–172.
4. Seijkens T. et al., 2013. CD40–CD40L: Linking pancreatic, adipose tissue and vascular inflammation in type 2 diabetes and its complications. Diabetes and Vascular Disease Research. 10: 115 - 122.
5. Daoussis D. et al., 2004. Targeting CD40L: a promising therapeutic approach. Clin Diagn Lab Immunol. 11(4):635-41.

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