KC-1098

293T-cyno-CD40L Cell Line

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Background of 293T-cyno-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-1098
Cell Line Name	293T-cyno-CD40L Cell Line
Host Cell Line	HEK293T
Description	HEK293T cell line stable expressing exogenous cyno 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 + 1 μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Fibroblastoid cells growing as monolayer
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 cyno CD40L cell line was generated using lentiviral vector expressing cyno CD40L sequence.

Characterization

Figure: Characterization of CD40L overexpressing in 293T stable clones using FACS.

Cell Resuscitation

1.Prewarm culture medium (DMEM supplemented with 10% FBS and 1 μg/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 9.0 mL complete culture medium.
5.Spin at ~ 125 x 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% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
2.Keep the freezing medium on ice and label cryovials.
3.Harvest cells to a sterile, conical centrifuge tube during the logarithmic growth period and count the cells.
4.Centrifuge the cells at 250 x 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 (April 1992). Identification of a novel surface protein on activated CD4+ T cells that induces contact-dependent B cell differentiation (help). The Journal of Experimental Medicine. 175 (4): 1091–101.
2.Lederman S, Yellin MJ, Inghirami G, Lee JJ, Knowles DM, Chess L (December 1992). Molecular interactions mediating T-B lymphocyte collaboration in human lymphoid follicles. Roles of T cell-B-cell-activating molecule (5c8 antigen) and CD40 in contact-dependent help. Journal of Immunology. 149 (12): 3817–26.
3.Lederman S, Yellin MJ, Cleary AM, Pernis A, Inghirami G, Cohn LE, Covey LR, Lee JJ, Rothman P, Chess L (March 1994). 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. Journal of Immunology. 152 (5): 2163–71.