KC-4349

293T-EGFR-B7H3-KO-2B3 Cell Line

47123

Home » 293T-EGFR-B7H3-KO-2B3 Cell Line

Background of 293T-EGFR-B7H3-KO-2B3 Cell Line

The immune checkpoint molecule B7-H3, also known as CD276, is a membrane protein member of the B7-CD28 family of immunomodulatory proteins, a type I membrane protein that is sequence-similar to the extracellular domain of programmed death receptor-1 (PD-L1). It is a newly identified immunoregulatory protein member of the B7 family, is an attractive and promising target for cancer immunotherapy because it is overexpressed in tumor tissues while showing limited expression in normal tissues and participating in tumor microenvironment (TME) shaping and development.

Specifications

Catalog Number	KC-4349
Cell Line Name	293T-EGFR-B7H3-KO-2B3 Cell Line
Host Cell Line	293T-EGFR-KO-1C2
Description	Stable 293T-EGFR-KO-1C2 clone with human B7H3 gene knockout, No.2B3
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	DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10% FBS
Selection Marker	N/A
Morphology	epithelial
Subculture	Split saturated culture 1:5-1:8 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

Cell Line Generation

293T-EGFR-B7H3-KO-2B3 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of 293T-EGFR-B7H3-KO-2B3 cell line stable clone using PCR sequencing.

Figure 2: Characterization of 293T-EGFR-B7H3-KO-2B3 cell line stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS)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 1:5-1:8 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

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.
Yang S, Wei W, Zhao Q. B7-H3, a checkpoint molecule, as a target for cancer immunotherapy. Int J Biol Sci. 2020 Mar 25;16(11):1767-1773. doi: 10.7150/ijbs.41105. PMID: 32398947; PMCID: PMC7211166.