KC-4712

CHOK1-rat-B7H3 Cell Line

48930

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Background of CHOK1-rat-B7H3 Cell Line

B7H3 aliases is CD276, CD276 (CD276 Molecule) is a Protein Coding gene. 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-4712
Cell Line Name	CHOK1-rat-B7H3 Cell Line
Host Cell Line	CHOK1
Description	CHOK1 cell line stably expressing exogenous rat-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 + 10 μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Fibroblastoid cells growing as monolayer
Subculture	Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-2 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 24 hours
Mycoplasma Status	Negative

Cell Line Generation

CHOK1-rat-B7H3 cell line was generated using a lentiviral vector expressing the rat B7H3 sequence.

Characterization

Figure 1: Figure 1: Characterization of rat B7H3 overexpressing in CHOK1 stable clones using FACS.

Figure2: Characterization of CHOK1-rat-B7H3 cell line stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 10 μg/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 9.0 mL 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 1-2 × 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. Jiang B, Zhang T, Liu F, Sun Z, Shi H, Hua D, Yang C. The co-stimulatory molecule B7-H3 promotes the epithelial-mesenchymal transition in colorectal cancer. Oncotarget. 2016 May 31;7(22):31755-71. doi: 10.18632/oncotarget.9035. PMID: 27145365; PMCID: PMC5077974.
2. Zhang P, Chen Z, Ning K, Jin J, Han X. Inhibition of B7-H3 reverses oxaliplatin resistance in human colorectal cancer cells. Biochem Biophys Res Commun. 2017 Aug 26;490(3):1132-1138. doi: 10.1016/j.bbrc.2017.07.001. Epub 2017 Jul 1. PMID: 28676400.