KC-2956

CHOK1-B7H4-Cell-Line

26841

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Background of CHOK1-B7H4-Cell-Line

B7H4, also named as B7x and B7S1, is transmembrane glycosylated protein belonging to B7 family of immunomodulatory proteins. B7H4 is expressed on the surface of many immune cells, including activated lymphocytes, macrophages, monocytes, dendritic cells, it can dampen T cells response and induces cell cycle arrest of T cells.

Specifications

Catalog Number	KC-2956
Cell Line Name	CHOK1-B7H4-Cell-Line
Description	Stable CHOK1 cell line expressing exogenous human B7H4 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-like
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

CHOK1 human B7H4 cell line was generated using a retroviral vector expressing the human B7H4 sequence.

Characterization

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% RPMI-1640 + 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

Sica, Gabriel L, In-Hak Choi, Gefeng Zhu, Koji Tamada, Sheng-Dian Wang, Hideto Tamura, Andrei I Chapoval, Dallas B Flies, J°îrgen Bajorath, and Lieping Chen. 2003. ¡ùB7-H4, a Molecule of the B7 Family, Negatively Regulates T Cell Immunity.¡ì Immunity 18 (6): 849Ã¿61. doi:10.1016/S1074-7613(03)00152-3.
Prasad, Durbaka V R, Sabrina Richards, Xoi Muoi Mai, and Chen Dong. 2003. ¡ùB7S1, a Novel B7 Family Member That Negatively Regulates T Cell Activation.¡ì Immunity 18 (6): 863Ã¿73. doi:10.1016/S1074- 7613(03)00147-X.
Podojil, Joseph R, and Stephen D Miller. 2017. ¡ùPotential Targeting of B7-H4 for the Treatment of Cancer.¡ì Immunological Reviews 276 (1). Wiley/Blackwell (10.1111): 40Ã¿51. doi:10.1111/imr.12530.