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HEK-Blue™ CD40L Cells

HEK-Blue™ CD40L cells Unit size Cat. code Docs Qty Price
3-7 x 10e6 cells
hkb-cd40
+-
$1,142.00

CD40L Sensor Cells

HEK-Blue™ CD40L cells can serve to measure the bioactivity of CD40L through the secretion of embryonic alkaline phosphatase (SEAP) upon NF-κB activation following CD40 stimulation. These cells were generated by stable transfection of HEK293 cells with the human CD40 gene and an NF-κB-inducible SEAP construct. 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 using QUANTI-Blue™. 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. IL-1β- and TNF-α-mediated SEAP production can be blocked using neutralizing antibodies, such as anti-hIL-1β-IgG and anti-hTNF-α-IgA  respectively.


Stimulation of HEK-Blue™ CD40L cells by recombinant human CD40L. After a 24 h incubation, SEAP activity was assessed using QUANTI-Blue™ and reading the optical density (O.D.) at 655 nm.

Ligand EC50 Response Ratio
CD40 20 +/- 10 ng/ml 25

Note: The response ratio was calculated by dividing the OD at 655 nm for the treated cells by the OD at 655 nm for the untreated cells.


HEK-Blue™ CD40L cells were stimulated with various human recombinant cytokines; IFNα (1000 IU/ml), IFNβ (1000 IU/ml), IFNγ (1000 IU/ml), IL-1β (100 ng/ml), IL-4 (100 ng/ml), IL-6 (100 ng/ml), IL-13 (100 ng/ml), IL-18 (100 ng/ml), TGF-β (10 ng/ml), TNF-α (100 ng/ml) and CD40L (100 ng/ml). After a 24 h incubation, SEAP activity was assessed using QUANTI-Blue™ and reading the O.D. at 655 nm.

Neutralization of CD40L response
Neutralization of CD40L response

Anti-hCD40L-IgA was incubated with 5 ng/ml of hCD40L for 30 minutes prior to the addition of HEK-Blue™ CD40L cells, which were incubated with the antibody and cytokine for a further 24 h. Levels of SEAP in the supernatant were measured using QUANTI-Blue™ and reading the O.D. at 655 nm.

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Specifications

Antibiotic resistance: blasticidin, Zeocin™

Growth medium: DMEM, 4.5 g/l glucose, 2-4 mM L-glutamine, 10% (v/v) fetal bovine serum, 50 U/ml penicillin, 50 μg/ml streptomycin, 100 μg/ml Normocin™

Guaranteed mycoplasma-free

Shipped on dry ice

Screen for molecules that interfere with CD40L-CD40 cross-talk

Detection range for human CD40L: 5 ng - 1 μg/ml

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Contents

• 1 vial containing 3-7 x 106 cells

• 100 μl Blasticidin (10 mg/ml)

• 100 μl Zeocin™ (100 mg/ml)

• 1 ml Normocin™ (50 mg/ml)

• 1 pouch of QUANTI-Blue™ (SEAP detection medium)

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Description

HEK-Blue CD40L pathway

CD40 ligand (CD40L) is a member of the tumor necrosis factor (TNF) family of cell surface interaction molecules. It is mainly expressed in CD4+ T cells and interacts with CD40 on antigen-presenting cells to regulate both humoral and cellular immune responses. 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.

In HEK-Blue™ CD40L cells, binding of CD40L to its receptor CD40 triggers a signaling cascade leading to the activation of NF-κB and the subsequent production of SEAP.

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Citations

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