KC-4394

Raji-EGFR-B7H3-Cell-Line

46363

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Background of Raji-EGFR-B7H3-Cell-Line

Epidermal Growth Factor Receptor (EGFR) is a transmembrane glycoprotein that constitutes one of the four members of ErbB family of tyrosine kinase receptors. Activation of EGFR leads to autophosphorylation of receptor tyrosine kinase that initiates a cascade of downstream signaling pathways involved in regulating cellular proliferation, differentiation, and survival. EGFR is abnormally activated by various mechanisms like receptor overexpression, mutation, ligand-dependent receptor dimerization, ligand-independent activation and is associated with the development of variety of human cancers. 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). It is a newly identified immunoregulatory protein member of the B7 family, is an attractive and promising target for cancer immunotherapy because it is overexpressed in tumor tissues while showing limited expression in normal tissues and participating in tumor microenvironment (TME) shaping and development.

Specifications

Catalog Number	KC-4394
Cell Line Name	Raji-EGFR-B7H3-Cell-Line
Host Cell Line	Raji
Description	Stable Raji cell line expressing exogenous human EGFR 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% RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640 + 10% FBS + 2μg/mL Puromycin+ 400µg/mL Hygromycin B
Selection Marker	Puromycin, Hygromycin B
Morphology	Mostly single, round (some polymorph) cells in suspension
Subculture	Split saturated culture 1:3 every 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 24-36 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

Raji-EGFR-B7H3 cell line was generated using a lentiviral vector expressing the human EGFR B7H3 sequence.

Characterization

Figure 1: Characterization of EGFR B7H3 overexpression in Raji stable clones using FACS.

Figure 2: Characterization of EGFR B7H3 in the Raji stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS + 2μg/mL Puromycin+ 400µg/mL Hygromycin B)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 every 3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 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

Singh D, Attri BK, Gill RK, Bariwal J. Review on EGFR Inhibitors: Critical Updates. Mini Rev Med Chem. 2016;16(14):1134-66. doi: 10.2174/1389557516666160321114917. PMID: 26996617.
Zhou WT, Jin WL. B7-H3/CD276: An Emerging Cancer Immunotherapy. Front Immunol. 2021 Jul 19;12:701006. doi: 10.3389/fimmu.2021.701006. PMID: 34349762; PMCID: PMC8326801.
Yang S, Wei W, Zhao Q. B7-H3, a checkpoint molecule, as a target for cancer immunotherapy. Int J Biol Sci. 2020 Mar 25;16(11):1767-1773. doi: 10.7150/ijbs.41105. PMID: 32398947; PMCID: PMC7211166.