KC-0218

293T-2Ig-B7H3-Cell-Line

20035

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Background of 293T-2Ig-B7H3-Cell-Line

CD276, also named as human B7 homolog 3 (B7-H3), is a member of the B7 immune protein family. CD276 is expressed on dendritic cells, T cells, B cells, and NK cells, and play an important role immune cell activation as a costimulatory molecule.

Specifications

Catalog Number	KC-0218
Cell Line Name	293T-2Ig-B7H3-Cell-Line
Host Cell Line	Human HEK293T cell line
Description	Stable HEK293T cell line expressing exogenous human B7H3 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+0.5µg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:3-1:6 every 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 human B7H3 cell line was generated using a lentiviral vector expressing the human B7H3 sequence.

Characterization

Figure: Characterization of B7H3 overexpression in 293T stable clones using FACS

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS + 0.5μg/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 1:3-1:6 every 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

Chapoval AI, Ni J, Lau JS, Wilcox RA, Flies DB, Liu D, Dong H, Sica GL, Zhu G, Tamada K, Chen L (March 2001). "B7-H3: a costimulatory molecule for T cell activation and IFN-gamma production". Nature Immunology.
2 (3): 269ÿ74 2. Kontos F, Michelakos T, Kurokawa T, Sadagopan A, Schwab JH, Ferrone CR, Ferrone S (October 2020). "B7-H3: an attractive target for antibody-based immunotherapy". Clinical Cancer Research: clincanres.2584.2020