KC-4963

CHOK1-rat-CD40L Cell Line

53915

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Background of CHOK1-rat-CD40L Cell Line

CD40LG (CD40 Ligand) CD154, also known as CD40 ligand, is a costimulatory molecule involved in humoral and adaptive immune responses upon pairing with its classical receptor, CD40. it is a Protein Coding gene. CD154 (CD40-ligand) is a critical immune regulator. CD154 expression is tightly regulated and largely restricted to activated CD4 T cells. Diseases associated with CD40LG include Immunodeficiency With Hyper-Igm, Type 1 and Cd40 Ligand Deficiency. The CD154/CD40 dyad is a key participant in the pathogenesis of many autoimmune diseases, including systemic lupus erythematosus (SLE).

Specifications

Catalog Number	KC-4963
Cell Line Name	CHOK1-rat-CD40L Cell Line
Host Cell Line	CHOK1
Description	Stable CHOK1 cell line expressing exogenous rat CD40L gene
Quantity	One vial 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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS + 10μg/ml Puromycin
Selection Marker	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 24 hours
Mycoplasma Status	Negative

Cell Line Generation

CHOK1-rat-CD40L Cell Line was generated using a lentiviral vector expressing the rat CD40L sequence.

Characterization

Figure 1: Characterization of rat CD40L overexpression in the CHOK1 rat CD40L stable clone using FACS.

Figure 2: Characterization of rat CD40L and its mutants overexpressing in CHOK1 stable clones using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 10μg/mL Puromycin)in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO₂ incubator.
8. Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage.

References

1. Brunner M, Zhang M, Genin A, Ho IC, Cron RQ. A T-cell-specific CD154 transcriptional enhancer located just upstream of the promoter. Genes Immun. 2008 Oct;9(7):640-9. doi: 10.1038/gene.2008.67. Epub 2008 Aug 21. PMID: 18719603; PMCID: PMC7333362.
2. Yang Q, Ji G, Li J. STEAP2 is down-regulated in breast cancer tissue and suppresses PI3K/AKT signaling and breast cancer cell invasion in vitro and in vivo. Cancer Biol Ther. 2020;21(3):278-291. doi: 10.1080/15384047.2019.1685290. Epub 2019 Nov 7. PMID: 31696760; PMCID: PMC7012168.
3. Allard CC, Salti S, Mourad W, Hassan GS. Implications of CD154 and Its Receptors in the Pathogenesis and Treatment of Systemic Lupus Erythematosus. Cells. 2024 Sep 28;13(19):1621. doi: 10.3390/cells13191621. PMID: 39404385; PMCID: PMC11482534.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.