KC-3930

293T-B7H3-KO-cyno-2Ig-B7H3 Cell Line

40328

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

B7H3 is an immune checkpoint molecule in the epithelial mesenchymal transition (EMT) pathway. B7H3 plays an important role in cell proliferation, invasion and migration of malignant tumors, and its abnormal expression is related to the occurrence of tumors. This finding may become a new target for the treatment of malignant tumors.

Specifications

Catalog Number	KC-3930
Cell Line Name	293T-B7H3-KO-cyno-2Ig-B7H3 Cell Line
Host Cell Line	293T-B7H3-KO
Description	Stable 293T-B7H3-KO cell line expressing exogenous cyno 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 +1ug/ml Puromycin
Selection Marker	Puromycin
Morphology	Fibroblastoid cells growing as a monolayer
Subculture	Split saturated culture 1:4-1:5 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-B7H3-KO-cyno-2Ig-B7H3 cell linewas generated using a lentiviral vector expressing the cyno B7H3 sequence.

Characterization

Figure 1: Characterization of cyno B7H3 overexpression in 293T-B7H3-KO stable clones using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 1 μg/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 1:4-1:5 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

Liu S, Liang J, Liu Z, Zhang C, Wang Y, Watson AH, Zhou C, Zhang F, Wu K, Zhang F, Lu Y, Wang X. The Role of CD276 in Cancers. Front Oncol. 2021 Mar 26;11:654684. doi: 10.3389/fonc.2021.654684. PMID: 33842369; PMCID: PMC8032984.
Zhou WT, Jin WL. B7-H3/CD276: An Emerging Cancer Immunotherapy. Front Immunol. 2021 Jul 19;12:701006. doi: 10.3389/fimmu.2021.701006. PMID: 34349762; PMCID: PMC8326801.