膜杰作
Star Staining
描述（Description）
The Human CD40 (Luc) HEK293 Reporter Cell was engineered to not only express NF-κB signaling response element, but also express the receptor full length human CD40 (Gene ID: 958), which can drive luciferase expressing systems by CD40 ligand/ agonist antibody stimulation. In the absence of agonist antibody or CD40 ligand, the CD40 receptor is not activated and luminescence signal is low. In the presence of agonist antibody or CD40 ligand, the CD40 pathway-activated luminescence can be detected in a dose-dependent manner. This reporter cell can also be used to test agonist antibody whether in an FcγR-dependent manner to strengthen the agonistic activity.
应用说明（Application）
• Screen for ligands or agonist antibodies that can bind and activate CD40.
生长特性（Growth Properties）
Adherent
筛选标记（Selection Marker）
Puromycin (2 μg/mL) + Hygromycin (20 μg/mL)
培养基（Culture Medium）
DMEM medium + 10% FBS
冻存液（Freeze Medium）
Serum-free cell cryopreservation medium
装量（Quantity）
1 vial contains at least 5×10^6 cells in 1 mL serum-free cryopreservation medium
存储（Storage）
Frozen in liquid nitrogen.
支原体检测（Mycoplasma Testing）
Negative
无菌检测（Sterility Testing）
Negative
使用说明（Instructions for Use）
See data sheet for detailed culturing and assay protocol.
Receptor Assay
Expression analysis of human CD40 on Human CD40 (Luc) HEK293 Reporter Cell by FACS.
Cell surface staining was performed on Human CD40 (Luc) HEK293 Reporter Cell or negative control cell using PE-labeled anti-human CD40 antibody.
Protocol
Application
Agonistic activity analysis of anti-human CD40 antibody.
This reporter cell was incubated with serial dilutions of antibodies in the presence of CHO or CHO/CD32b. Sotigalimab could depend on CD32b-mediated crosslinking to strengthen CD40 signaling. The EC50 of Sotigalimab in the presence of CHO/CD32b was approximately 0.079 μg/mL.
Protocol
Inhibition of human CD40 ligand protein-induced reporter activity by anti-human CD40 ligand neutralizing antibody.
This reporter cell was incubated with serial dilutions of antibodies in the presence of human CD40 ligand protein with a final concentration of 0.1 μg/mL. The EC50 of anti-human CD40 ligand neutralizing antibody (Frexalimab) was approximately 0.26 μg/mL.
Protocol
Signaling Bioassay
Response to anti-human CD40 antibody (RLU).
The Human CD40 (Luc) HEK293 Reporter Cell was stimulated with serial dilutions of anti-human CD40 antibody. The EC50 was approximately 0.096 μg/mL.
Protocol
Response to anti-human CD40 antibody (FOLD).
The Human CD40 (Luc) HEK293 Reporter Cell was stimulated with serial dilutions of anti-human CD40 antibody. The max induction fold was approximately 14.
Protocol
Response to human CD40 ligand protein (RLU).
The Human CD40 (Luc) HEK293 Reporter Cell was stimulated with serial dilutions of human CD40 ligand protein. The EC50 was approximately 0.024 μg/mL.
Protocol
Response to human CD40 ligand protein (FOLD).
The Human CD40 (Luc) HEK293 Reporter Cell was stimulated with serial dilutions of human CD40 ligand protein. The max induction fold was approximately 9.16.
Protocol
Passage Stability
Passage stability analysis by Signaling Bioassay.
The continuously growing Human CD40 (Luc) HEK293 Reporter Cell was stimulated with serial dilutions of anti-human CD40 antibody. Anti-human CD40 antibody stimulated response demonstrates passage stabilization (fold induction and EC50) across passage 6-22.
Protocol
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背景（Background）
CD40 is also known as TNFRSF5, Bp50, CDW40, MGC9013, TNFRSF5 and p50, is a member of the TNF receptor superfamily which are single transmembrane-spanning glycoproteins, and plays an essential role in mediating a broad variety of immune and inflammatory responses including T cell-dependent immunoglobulin class switching, memory B cell development, and germinal center formation. CD40 is a costimulatory protein found on antigen presenting cells and is required for their activation. The binding of CD154 (CD40L) on TH cells to CD40 activates antigen presenting cells and induces a variety of downstream effects. CD40 contains 4 cysteine-rich repeats in the extracellular domain, and is expressed in B cells, dendritic cells, macrophages, endothelial cells, and several tumor cell lines. The extracellular domain has the cysteinerich repeat regions, which are characteristic for many of the receptors of the TNF superfamily. Interaction of CD40 with its ligand, CD40L, leads to aggregation of CD40 molecules,which in turn interact with cytoplasmic components to initiate signaling pathways. Early studies on the CD40-CD40L system revealed its role in humoral immunity. Defects in CD40 result in hyper-IgM immunodeficiency type 3 (HIGM3), an autosomal recessive disorder characterized by an inability of B cells to undergo isotype switching, as well as an inability to mount an antibody-specific immune response, and a lack of germinal center formation.
Permits & Restrictions
This cell line is provided for research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. You are not allowed to share, distribute, sell, modify, sublicense, or otherwise make this cell line available for use to other laboratories, departments, research institutions, hospitals, universities, or biotech companies. AcroBiosystems does not warrant the suitability of this cell line for any particular use, and does not accept any liability in connection with the handling or use of this cell line.
