KC-1002

CT26.WT-human-B7H4 Cell Line

21361

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Background of CT26.WT-human-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-1002
Cell Line Name	CT26.WT-human-B7H4 Cell Line
Host Cell Line	CT26
Description	CT26.WT cell line stable 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
Subculture	Split saturated culture 1:4~1:10 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	Yes

Cell Line Generation

CT26.WT-B7H4 Cell Line was generated using lentivirus expressing cynomolgus B7H4 gene sequence.

Characterization

Figure 1: Characterization of B7H4 overexpressing in CT26.WT stable clones using FACS.

Cell Resuscitation

Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 10ug/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 x 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 x 10

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 x g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3 x106 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

1. Sica, Gabriel L, In-Hak Choi, Gefeng Zhu, Koji Tamada, Sheng-Dian Wang, Hideto Tamura, Andrei I Chapoval, Dallas B Flies, Jürgen Bajorath, and Lieping Chen. 2003. “B7-H4, a Molecule of the B7 Family, Negatively Regulates T Cell Immunity.” Immunity 18 (6): 849–61. doi:10.1016/S1074-7613(03)00152-3.
2.Prasad, Durbaka V R, Sabrina Richards, Xoi Muoi Mai, and Chen Dong. 2003. “B7S1, a Novel B7 Family Member That Negatively Regulates T Cell Activation.” Immunity 18 (6): 863–73. doi:10.1016/S1074- 7613(03)00147-X.
3.Podojil, Joseph R, and Stephen D Miller. 2017. “Potential Targeting of B7-H4 for the Treatment of Cancer.” Immunological Reviews 276 (1). Wiley/Blackwell (10.1111): 40–51. doi:10.1111/imr.12530.