KC-1286

CHOK1-CD40-Cell-Line

21861

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Background of CHOK1-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-1286
Cell Line Name	CHOK1-CD40-Cell-Line
Host Cell Line	Chinese hamster ovary CHO-K1 cell line
Description	CHOK1 cell line stable expressing exogenous human 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% 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 1-2 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 24 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

293T human CD40 cell line was generated using lentiviral vector expressing human CD40 sequence.

Characterization

Figure: Characterizatton of CD40 overexpressing in CHOK1 stable clones using FACS.

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS + 10µg/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 1:4-1:8 every 2-3 days; seed out at about 1-2 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 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): 167ÿ178.
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): 474ÿ83.
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): 697ÿ706.