CD40L Reporter HEK 293 Cells

CD40L Reporter Cells

HEK-Blue™ CD40L Cells signaling pathway

HEK-Blue™ CD40L cells were engineered from the human embryonic kidney HEK 293 cell line to measure the bioactivity of CD40L through the secretion of embryonic alkaline phosphatase (SEAP) upon NF-κB activation. CD40 Ligand (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 [1-3].

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description:

HEK-Blue™ CD40L cells were generated by stable expression of the human CD40 gene and an NF-κB-inducible SEAP construct. In these cells, the binding of CD40L to its receptor CD40 triggers a signaling cascade leading to the activation of NF-κB and the subsequent production of SEAP. CD40L-CD40 interaction can be monitored by assessing the levels of SEAP in the supernatant using QUANTI-Blue™ Solution, a detection reagent.

Of note, HEK293 cells express endogenously the receptors for the cytokines IL-1β and TNF-α which share a common signaling pathway with CD40L. Consequently, HEK-Blue™ CD40L cells also respond to IL-1β and TNF-α. However, the IL-1β- and TNF-α-mediated SEAP production can be blocked using neutralizing antibodies, such as anti-hIL-1β-IgG and anti-hTNF-α-IgA2 respectively.

Key Features:

• Fully functional CD40L signaling pathway
• Readily assessable SEAP reporter activity

Applications:

• Detection of human and murine CD40L
• Screening of anti-CD40 and anti-CD40L antibodies

References:

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.

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

Antibiotic resistance: Blasticidin, Zeocin®

Growth medium: DMEM, 4.5 g/l glucose, 2 mM L-glutamine, 10% (v/v)  heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml Normocin™

Guaranteed mycoplasma-free

Specification: human and murine CD40L

Detection range:

• 3 ng - 1 μg/ml for human CD40L
• 3  ng - 1 μg/ml for murine CD40L

This product is covered by a Limited Use License (See Terms and Conditions).

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 and some areas in Asia)

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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