KC-4181

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

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Background of 293T-B7H3-KO-cyno-B7H3-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). In normal human tissues, the expression level of B7-H3 is low, but in a variety of tumor cancers, B7-H3 is abnormally high, which plays an important role in the development of tumors, immune escape and other processes, and is associated with the poor prognosis of tumors. At present, there are a total of 109 biologics developed for B7-H3 targets, but only 33 are in the clinical stage, of which ADC and CAR-T therapy are the main ones.

Specifications

Catalog Number	KC-4181
Cell Line Name	293T-B7H3-KO-cyno-B7H3-Cell-Line
Host Cell Line	293T-B7H3-KO
Description	Stable 293T-B7H3-KO clone 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 + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:3-1:6 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-B7H3 cell line was generated using a lentiviral vector expressing the cyno-B7H3 sequence.

Characterization

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

Figure 2: Characterization of cyno B7H3 in the 293T B7H3-KO-cyno-B7H3 stable clone 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 1:3-1:6 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

Dong P, Xiong Y, Yue J, Hanley SJB, Watari H. B7H3 As a Promoter of Metastasis and Promising Therapeutic Target. Front Oncol. 2018 Jul 6;8:264. doi: 10.3389/fonc.2018.00264. PMID: 30035102; PMCID: PMC6043641.