KC-1667

293T-NFκB-Luc2-mouse-CD40-reporter-Cell-Line

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Background of 293T-NFκB-Luc2-mouse-CD40-reporter-Cell-Line

CD40, the receptor for CD40L, is a member of the TNF receptor (TNFR) superfamily. It is expressed in a plethora of cell types, including B cells, macrophages and dendritic, endothelial and epithelial cells.The CD40/CD40L interactions play a central role in orchestrating the immune response. CD40L is produced by both Th1 and Th2 helper T cells, as well as by mast cells, basophils and eosinophils.Signals induced by CD40L expressed in Th1 and Th2 cells are required for macrophage and B‐cell activation, respectively.It transduces TRAF6- and MAP3K8-mediated signals that activate ERK in macrophages and B cells, leading to induction of immunoglobulin secretion.

Specifications

Catalog Number	KC-1667
Cell Line Name	293T-NFκB-Luc2-mouse-CD40-reporter-Cell-Line
Host Cell Line	293T-NFκB-Luc2
Description	Stable 293T cell line expressing exogenous luciferase gene under the control of CD40 signaling pathway.
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Cell model for monitoring mouse CD40 signaling pathway
Freezing Medium	70% DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10% FBS +150μg/ml Hygromycin B+1μg/ml puromycin
Selection Marker	Hygromycin B and Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

293T-NFκB-Luc2-mouse-CD40-reporter cell line was generated using lentivirus expressing mouse CD40 sequence.

Characterization

Figure 1. Characterization of mouse-CD40 overexpression in the 293T-NFκB-Luc2-mouse-CD40 stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS, 150μg/ml Hygromycin and 1μg/ml Puromycin)in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage

References

Eliopoulos AG, Wang CC, Dumitru CD, Tsichlis PN. Tpl2 transduces CD40 and TNF signals that activate ERK and regulates IgE induction by CD40. EMBO J. 2003 Aug 1;22(15):3855-64. doi: 10.1093/emboj/cdg386. PMID: 12881420; PMCID: PMC169059.
Karnell JL, Rieder SA, Ettinger R, Kolbeck R. Targeting the CD40-CD40L pathway in autoimmune diseases: Humoral immunity and beyond. Adv Drug Deliv Rev. 2019 Feb 15;141:92-103. doi: 10.1016/j.addr.2018.12.005. Epub 2018 Dec 13. PMID: 30552917.