KC-3684

Raji-B7H3 Cell Line

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Background of Raji-B7H3 Cell Line

B7-H3, also known as CD276. The protein encoded by this gene belongs to the immunoglobulin superfamily, and thought to participate in the regulation of T-cell-mediated immune response. Studies show that while the transcript of this gene is ubiquitously expressed in normal tissues and solid tumors, the protein is preferentially expressed only in tumor tissues. Additionally, it was observed that the 3' UTR of this transcript contains a target site for miR29 microRNA, and there is an inverse correlation between the expression of this protein and miR29 levels, suggesting regulation of expression of this gene product by miR29. Alternatively spliced transcript variants encoding different isoforms have been found for this gene.

Specifications

Catalog Number	KC-3684
Cell Line Name	Raji-B7H3 Cell Line
Clone Number	6#
Host Cell Line	Raji
Description	Stable Raji cell line expressing exogenous 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
Selection Marker	Puromycin
Morphology	Lymphoblast
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

Raji B7H3 Cell Line was generated using a lentiviral vector expressing the B7H3 sequence.

Characterization

Figure 1: Characterization of endogenous B7H3 expression in Raji cells using FACS.

Figure 2: Characterization of B7H3 overexpression in the Raji B7H3 stable clone using FACS.

Figure 3: Characterization of B7H3 overexpressing in Raji B7H3 stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 2μg/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% RPMI1640 + 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. Chen C, Wang Y, Zhong K, Jiang C, Wang L, Yuan Z, Nie C, Xu J, Guo G, Zhou L, Yang M, Tong A. Frequent B7-H3 overexpression in craniopharyngioma. Biochem Biophys Res Commun. 2019 Jun 25;514(2):379-385. doi: 10.1016/j.bbrc.2019.04.142. Epub 2019 Apr 28. PMID: 31043272.
2. Gootjes EC, Kraan J, Buffart TE, Bakkerus L, Zonderhuis BM, Verhoef C, Verheul HMW, Sleijfer AS. CD276-Positive Circulating Endothelial Cells Do Not Predict Response to Systemic Therapy in Advanced Colorectal Cancer. Cells. 2020 Jan 5;9(1):124. doi: 10.3390/cells9010124. PMID: 31948091; PMCID: PMC7016770.