KC-6546

293T-EGFR-B7H3-KO-dog-B7H3-HA-Low-Cell-Line

64150

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Background of 293T-EGFR-B7H3-KO-dog-B7H3-HA-Low-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-6546
Cell Line Name	293T-EGFR-B7H3-KO-dog-B7H3-HA-Low-Cell-Line
NCBI/UniProt Accession Number	A0A8P0SM01
Clone Number	17#
Host Cell Line	293T-EGFR-B7H3-KO
Description	Stable 293T-EGFR-B7H3-KO clone expressing exogenous dog-B7H3-HA gene in low level
Quantity	One vial 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

Cell Line Generation

293T-EGFR-B7H3-KO-dog-B7H3-HA-Low-cell-line was generated using a lentiviral vector expressing the dog-B7H3-HA sequence.

Characterization

Figure 1: Characterization of dog-B7H3-HA overexpression in the 293T-EGFR-B7H3-KO-dog-B7H3-HA-Low stable clone using FACS.

Figure 2: Characterization of dog-B7H3 in the 293T-EGFR-B7H3-KO-dog-B7H3-HA-Low stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 1μg/mL puromycin)in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO₂ incubator.
8. Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage.

References

1.Mortezaee K. B7-H3 immunoregulatory roles in cancer. Biomed Pharmacother. 2023 Jul;163:114890.
2. 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.
3. 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.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.