KC-3663

293T-4Ig-B7H3-Cell-Line

27041

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Background of 293T-4Ig-B7H3-Cell-Line

CD276, also named as human B7 homolog 3 (4Ig-B7H3), is a member of the B7 immune protein family. CD276 is expressed on dendritic cells, T cells, B cells, and NK cells, and plays an important role in immune cell activation as a co-stimulatory molecule.

Specifications

Catalog Number	KC-3663
Cell Line Name	293T-4Ig-B7H3-Cell-Line
Host Cell Line	293T
Description	Stable HEK293T cell line expressing exogenous human 4Ig-B7H3 gene
Quantity	One vial 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 1:4-1:8 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 human 4Ig-B7H3 cell line was generated using a lentiviral vector expressing the human 4Ig-B7H3 sequence.

Characterization

Figure1: Characterization of human B7H3 overexpression in 293T human B7H3 stable clones using FACS.

Figure2: Characterization of endogenous human B7H3 expression in 293T cells using FACS.

Figure3: Characterization of human B7H3 expression in 293T human B7H3 stable clones using PCR sequencing.

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 1:4-1:8 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

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):
269–74.
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.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.