KC-0229

293T-CD154 (CD40L) Cell Line

20057

Home » 293T-CD154 (CD40L) Cell Line

Background of 293T-CD154 (CD40L) Cell Line

CD154, also named as CD40L ligand, is protein of TNF superfamily, mainly expressed on activated T cell, functioned as CD40 ligand, plays critical in immune response, such as B cell maturation, macrophage activation.

Specifications

Catalog Number	KC-0229
Cell Line Name	293T-CD154 (CD40L) Cell Line
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous CD154 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 1:4-1:6 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 CD154 cell line was generated using a lentiviral vector expressing the CD154 sequence.

Characterization

Figure 1: Characterization of CD154 overexpression in the 293T CD154 stable clone using FACS.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 1μg/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 1:4-1:6 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% DMEM + 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. Lederman S, Yellin MJ, Krichevsky A, Belko J, Lee JJ, Chess L. Identification of a novel surface protein on activated CD4+ T cells that induces contact-dependent B cell differentiation (help). J Exp Med. 1992 Apr 1;175(4):1091-101. doi: 10.1084/jem.175.4.1091. PMID: 1348081; PMCID: PMC2119166.
2. Lederman S, Yellin MJ, Cleary AM, Pernis A, Inghirami G, Cohn LE, Covey LR, Lee JJ, Rothman P, Chess L. T-BAM/CD40-L on helper T lymphocytes augments lymphokine-induced B cell Ig isotype switch recombination and rescues B cells from programmed cell death. J Immunol. 1994 Mar 1;152(5):2163-71. PMID: 7907632.