KC-2424
293T-mouse-B7H3-Hgih Cell Line
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.