KC-0913

293T-Cyno-CD40-Cell-Line

43695

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Background of 293T-Cyno-CD40-Cell-Line

CD40, also named as TNFRSF5, is a transmembrane protein of the TNF receptor superfamily, expressed on B cells, DC cells, macrophage, monocytes and plates, CD40 functioned as a costimulatory molecule and mediated broad immune and inflammatory response.

Specifications

Catalog Number	KC-0913
Cell Line Name	293T-Cyno-CD40-Cell-Line
Host Cell Line	293T
Description	HEK293T cell line stable expressing exogenous cyno CD40 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 Cyno CD40 cell line was generated using a lentiviral vector expressing the cyno CD40 sequence.

Characterization

Figure: Characterization of cyno CD40 overexpressing in 293T stable clones 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

Kawabe, Tsutomu; Naka, Tetsuji; Yoshida, Kanji; Tanaka, Takashi; Fujiwara, Hiroshi; Suematsu, Sachiko; Yoshida, Nobuaki; Kishimoto, Tadamitsu; Kikutani, Hitoshi (1994). The immune responses in CD40-deficient mice: Impaired immunoglobulin class switching and germinal center formation. Immunity. 1 (3): 167–178.
Chatzigeorgiou A, Lyberi M, Chatzilymperis G, Nezos A, Kamper E (2009). CD40/CD40L signaling and its implication in health and disease. BioFactors (Oxford, England). 35 (6): 474–83.
Carlring J, Altaher HM, Clark S, Chen X, Latimer SL, Jenner T, Buckle AM, Heath AW (May 2011). CD154-CD40 interactions in the control of murine B cell hematopoiesis. Journal of Leukocyte Biology. 89 (5): 697–706.