Background:CD40, a TNF receptor superfamily member, was initially identified on B lymphocytes. Antigen presenting cells (APCs) such as monocytes, basophils, dendritic cells, have been found to express CD40. Additionally, CD40 has been identified in non-immune cells such as endothelial cells and epithelial cells. A wide variety of carcinoma cells over-express CD40 as well. Interaction with CD40 ligand (CD40L, CD154) on CD4+ T helper lymphocytes triggers the expression of intercellular adhesion molecule (ICAM) and other pro-inflammatory cytokines. CD40:CD40L signaling simultaneously increases activation of antigen-specific T cells. CD40 activates NF-κB-dependent signaling in response to lipopolysaccharide (LPS) found on Gram negative bacterial pathogens. Furthermore, agonistic CD40 monoclonal antibodies have been shown to activate antigen presenting cells (APC) and promote anti-tumor T-cell responses in addition to fostering cytotoxic myeloid cells with the potential to control cancer in the absence of T-cell immunity. Description:Recombinant HEK293 cell line expressing full length human CD40 (Tumor necrosis factor receptor superfamily member 5; TNFRSF5). Expression is confirmed by real-time qPCR and Western Blot. This NF-ĸB luciferase reporter construct is stably integrated into the genome. The firefly luciferase gene is controlled by 4 copies of NF-κB response element located upstream of the TATA promoter. Following activation by human CD40 ligand, NF-ĸB transcription factor binds to the DNA response elements to induce transcription of the luciferase gene. Synonym(s): NF-κB cell line, NFkappaB cell line, luciferase reporter, NF-kB cell line, NFkB cell line, CD40 cell line, cd40 luciferase reporter, CD40 HEK293 cell line Host Cell line: HEK293 Mycoplasma Testing: This cell line has been screened using the MycoAlert™ Mycoplasma Detection Kit (Lonza, Cat.#LT07-118) to confirm the absence of Mycoplasma contamination. MycoAlert Assay Control Set(Lonza, Cat. #LT07-518) was used as a positive control. Growth Media: Opti-MEM® (Thermo Fisher Cat. #31985088), 10% heat-inactivated FBS, 1X Penicillin/ Streptomycin (Thermo Fisher, Cat. #10378016), 100 µg/ml Hygromycin B (Thermo Fisher, Cat. #10687010) and 400µg/ml Geneticin®, G418 Sulfate (Thermo Fisher, Cat. #11811031). Culture Conditions: Frozen Cells: Prepare T-25 culture flask with 10 ml of pre-warmed growth medium. Quickly thaw cells in a 37°C water bath with constant and slow agitation. Clean the outside of the vial with 70% ethanol and immediately transfer the entire content to the culture media without Hygromycin B and G418. Avoid pipetting up and down, and gently rock the flask to distribute the cells. Incubate the cells in a humidified 37°C incubator with 5% CO2. 48 hours after incubation, change to fresh medium without Hygromycin B and G418, without disturbing the attached cells. Continue to change the medium every 2-3 days until cells reach desired confluency. If slow cell growth occurs during resuscitation, increase FBS to 15% for the first week of culture. Begin adding Hygromycin B and G418 to medium after the first passage. Subculture: When cells reached 90% confluency, remove the medium and GENTLY wash once with PBS (without Magnesium or Calcium). These cells are loosely adherent and detach easily so do not resuspend the PBS directly onto the cell surface. Treat cells with 2 ml of 0.25% trypsin/EDTA and incubate for 2-3 minutes at 37°C. After confirming cell detachment by light microscopy, add 10 ml pre-warmed medium and gently pipette up and down to dissociate cell clumps. Transfer cells to a 15 ml conical tube and centrifuge at 200 x g for 5 minutes. Remove the medium and re-suspend cells in 10 ml of pre-warmed growth medium. Dispense 5 ml of the cell suspension into a new T75 flask containing pre-warmed 15 ml media. Incubate cells in a humidified 37°C incubator with 5% CO2. Freeze cells in freezing medium (10% DMSO in FBS) when cells reach 90% confluency. Cells have been demonstrated to be stable for at least 15 passages; BPS recommends preparing frozen stocks so cells are not used beyond passage 20. Format: Each vial contains ~2 x 106 cells in 1mL of 10% DMSO in FBS. Instructions for use: See data sheet for detailed culturing and assay protocol. Storage / Stability: Immediately upon receipt, store vials in liquid nitrogen. Application(s): The CD40 NF-ĸB Reporter HEK293 Stable Cell Line is ideal for High Throughput Screening (HTS) to identify potential CD40 agonistic monoclonal antibodies and CD40-specific inhibitors. Reference(s): 1.Li G et.al. (2013) Human Genetics in Rheumatoid Arthritis Guides a High-Throughput Drug Screen of the CD40 Signaling Pathway. PLoS Genet. 9: e1003487.

2.Pontrelli P et.al. (2006) CD40L Proinflammatory and Profibrotic Effects on Proximal Tubular Epithelial Cells: Role of NF-κB and Lyn. J. Amer. Soc. Neph. 17: 627.

3.Lavorgna A et.al. (2014) A Critical Role for IL-17RB Signaling in HTLV-1 Tax-Induced NF-κB Activation and T-Cell Transformation. PLoS Path. 10: e1004418.

4.Moschonas A et.al. (2012) CD40 Stimulates a \"Feed-Forward” NF-ĸB- Driven Molecular Pathway That Regulates IFN-β Expression in Carcinoma Cells. J. Immunol. 188: 5521.

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License Disclosure:Purchase of this cell line grants you with a 10-year license to use this cell line in your immediate laboratory, for research use only. This license does not permit you to share, distribute, sell, sublicense, or otherwise make the cell line available for use to other laboratories, departments, research institutions, hospitals, universities, or biotech companies. The license does not permit use of this cell line in humans or for therapeutic or drug use. The license does not permit modification of the cell line in any way. Inappropriate use or distribution of this cell line will result in revocation of the license and result in an immediate cease of sales and distribution of BPS products to your laboratory. BPS does not warrant the suitability of the cell line for any particular use, and does not accept any liability in connection with the handling or use of the cell line. Modifications of this cell line, transfer to another facility, or commercial use of the cells may require a separate license and additional fees; contact sales@bpsbioscience.com for details. Publications using this cell line should reference BPS Bioscience, Inc., San Diego.

Warning(s): Avoid freeze/thaw cycles. Scientific Category: Immunotherapy Product Type: Cell Line Data shown is lot-specific. Contact us for specific information on other lots.

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