KC-4087

293T-B7H3-KO Cell Line

40632

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Background of 293T-B7H3-KO Cell Line

"B7H3 (also known as CD276) is a type I transmembrane co-stimulatory/inhibitory molecule of the B7 family, which was firstly reported by Chen et al., in 2001. B7H3 is expressed in low levels in most normal cells, such as immune cells, osteoblasts, and endothelial cells, while it is overexpressed in a wide variety of malignant cells . The receptor of B7H3 is yet to be identified, so also is its role in immune regulation and inflammatory response still controversial. However, recent studies showed that it plays a regulatory role in immunosuppression. Besides its identified function in modulating immunity, B7H3 also plays a nonimmunologic role by regulating the proliferation, migration, invasion and metabolism of various cancer cells , and in osteoblastic differentiation and bone mineralization. "

Specifications

Catalog Number	KC-4087
Cell Line Name	293T-B7H3-KO Cell Line
Host Cell Line	293T
Description	Stable 293T cell line with B7H3 gene knockout, No.2B4
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
Selection Marker	NA
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-B7H3-KO cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of 293T-B7H3-KO Cell Line stable clone using PCR sequencing.

Figure 2: Characterization of 293T-B7H3-KO 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 1:4-1:8 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

Chen G, Tu Y, Aladelusi TO, Zhao S, Chen J, Jin L, Zhu D. Knocking down B7H3 expression enhances cell proliferation of SHEDs via the SHP1/AKT signal axis. Biochem Biophys Res Commun. 2020 Oct 20;531(3):282 289. doi: 10.1016/j.bbrc.2020.06.154. Epub 2020 Aug 14. PMID: 32800541.