KC-2424

293T-mouse-B7H3-Hgih Cell Line

24032

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Background of 293T-mouse-B7H3-Hgih Cell Line

B7H3, also known as CD276, is a type I transmembrane protein which belongs to the B7 superfamily of immune checkpoint molecules. B7-H3 is an immune checkpoint molecule that inhibits tumor antigen-specific immune responses. B7-H3 is present at low levels in most normal tissues but is overexpressed in a wide variety of cancers, including bladder, breast, cervical, colorectal, esophageal, glioma, kidney, liver, lung, ovarian, pancreatic, prostate, intrahepatic cholangiocarcinoma, liver, oral squamous cell carcinoma, endometrial cancer, and squamous cell carcinoma and gastric cancer, glioma, and melanoma. B7-H3 also possesses non-immunological pro-tumorigenic functions such as promoting migration, invasion, angiogenesis, chemoresistance, epithelial-to-mesenchymal transition, and affecting tumor cell metabolism.

Specifications

Catalog Number	KC-2424
Cell Line Name	293T-mouse-B7H3-Hgih Cell Line
NCBI/UniProt Accession Number	NM_001424679.1
Clone Number	NA
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous mouse-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 1:4-1:8 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-mouse-B7H3-High cell line was generated using lentivirus expressing mouse-B7H3 sequence.

Characterization

Figure 1. Characterization of mouse-B7H3 over-expression in the 293T-mouse-B7H3-High stable clone using FACS.

Figure 2. Characterization of mouse-B7H3 over-expression in the 293T-mouse-B7H3-High stable clone using PCR.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS, 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

Mortezaee K. B7-H3 immunoregulatory roles in cancer. Biomed Pharmacother. 2023 Jul;163:114890. doi: 10.1016/j.biopha.2023.114890. Epub 2023 May 15. PMID: 37196544.
Harland N, Maurer FB, Abruzzese T, Bock C, Montes-Mojarro IA, Fend F, Aicher WK, Stenzl A, Amend B. Elevated Expression of the Immune Checkpoint Ligand CD276 (B7-H3) in Urothelial Carcinoma Cell Lines Correlates Negatively with the Cell Proliferation. Int J Mol Sci. 2022 Apr 29;23(9):4969. doi: 10.3390/ijms23094969. PMID: 35563359; PMCID: PMC9100984.
Guo C, Figueiredo I, Gurel B, Neeb A, Seed G, Crespo M, Carreira S, Rekowski J, Buroni L, Welti J, Bogdan D, Gallagher L, Sharp A, Fenor de la Maza MD, Rescigno P, Westaby D, Chandran K, Riisnaes R, Ferreira A, Miranda S, Calì B, Alimonti A, Bressan S, Nguyen AHT, Shen MM, Hawley JE, Obradovic A, Drake CG, Bertan C, Baker C, Tunariu N, Yuan W, de Bono JS. B7-H3 as a Therapeutic Target in Advanced Prostate Cancer. Eur Urol. 2023 Mar;83(3):224-238. doi: 10.1016/j.eururo.2022.09.004. Epub 2022 Sep 13. Erratum in: Eur Urol. 2023 Jun;83(6):e168-e169. PMID: 36114082.