KC-2452
293T-mouse-CD40L-Middle Cell Line
Background of 293T-mouse-CD40L-Middle Cell Line
CD40L, also known CD154, Tumor Necrosis Factor Ligand Superfamily Member 5 (TNFSF5), gp39, and CD40L, is a gene that encodes the CD40 ligand protein. This crucial molecule belongs to the tumor necrosis factor (TNF) superfamily and plays a pivotal role in immune regulation and cellular interactions. The CD40L gene is primarily expressed on the surface of activated CD4+ T helper cells. Upon activation, CD40L binds to its cognate receptor CD40, which is present on antigen-presenting cells such as B cells, dendritic cells, monocytes/macrophages, and other non-hematopoietic cells like endothelial and epithelial cells. Disruptions or dysregulation of CD40L expression are associated with several diseases including XHIGM, autoimmune disorders, cardiovascular disease, and cancer, implicating its critical role in immune function and inflammation processes across diverse pathologies. There are multiple drugs targeting this target that are currently in Phase III clinical trials.
Specifications
| Catalog Number | KC-2452 |
|---|---|
| Cell Line Name | 293T-mouse-CD40L-Middle Cell Line |
| NCBI/UniProt Accession Number | NM_000074.3 |
| Host Cell Line | 293T |
| Description | Stable 293T cell line expressing exogenous mouse-CD40L gene |
| Quantity | Two vials of frozen cells (≥2-106/vial) |
| Stability | Stable in culture over a minimum of 10 passages |
| Application | Drug screening and biological assays |
| Freezing Medium | 70% DMEM + 20% FBS + 10% DMSO |
| Propagation Medium | DMEM + 10% FBS + 1μg/mL Puromycin |
| Selection Marker | Puromycin |
| Morphology | Epithelial |
| Subculture | Split saturated culture 1:4-1:5 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-mouse-CD40L-Middle cell line was generated using a lentiviral vector expressing the mouse-CD40L sequence.
Characterization
Figure: Characterization of mouse-CD40L overexpression in 293T stable clone using FACS.
Cell Resuscitation
- Prewarm culture medium (DMEM supplemented with 10% FBS 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 1:4-1:5 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
- Tang T, Cheng X, Truong B, Sun L, Yang X, Wang H. Molecular basis and therapeutic implications of CD40/CD40L immune checkpoint. Pharmacol Ther. 2021 Mar;219:107709. doi: 10.1016/j.pharmthera.2020.107709. Epub 2020 Oct 20. PMID: 33091428; PMCID: PMC7886970.
- Elgueta R, Benson MJ, de Vries VC, Wasiuk A, Guo Y, Noelle RJ. Molecular mechanism and function of CD40/CD40L engagement in the immune system. Immunol Rev. 2009 May;229(1):152-72. doi: 10.1111/j.1600-065X.2009.00782.x. PMID: 19426221; PMCID: PMC3826168.
- 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.