KC-2653

CHOK1 human 2Ig-B7H3 Cell Line

Home » CHOK1 human 2Ig-B7H3 Cell Line

Background

B7H3, also known as CD276, is a checkpoint in the B7 family that plays an immune regulatory role in T cell responses. It is abnormally and consistently expressed in several human cancers, and its overexpression is associated with weak prognosis. B7-H3 is expressed on many cells and is a driving factor for immune evasion. B7-H3 can serve as a target for blocking monoclonal antibodies (mAbs), combination therapy, and modifying chimeric antigen receptors.

Specifications

Catalog Number:
KC-2653
Cell Line Name:
CHOK1 human 2Ig-B7H3 Cell Line
Host Cell Line:
CHOK1
Description:
Stable CHOK1 cell line expressing exogenous human-2Ig-B7H3 gene
Quantity:
One vial of frozen cells (5×106 per vial)
Stability:
Stable in culture over a minimum of 10 passages
Application:
Drug screening and biological assays
Freeze Medium:
70% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium:
RPMI1640 + 10% FBS + 10μg/ml Puromycin
Selection Marker:
Puromycin
Morphology:
Epithelial
Subculture:
Split saturated culture 1:4-1:5 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 24 hours
Mycoplasma Status:
Negative

Cell Line Generation

CHO-K1 human-2Ig-B7H3 Cell Line was generated using a lentiviral vector expressing the human-2Ig-B7H3 sequence.

Characterization

Figure 1: Characterization of 2Ig-B7H3 overexpression in the CHO-K1 human-2Ig-B7H3 stable clone using FACS.
Figure 2: Characterization of 2Ig-B7H3 overexpression in the CHO-K1 human-2Ig-B7H3 stable clone using PCR sequence.

Application

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 10μg/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 1:4-1:5 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. 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.
2. Picarda E, Ohaegbulam KC, Zang X. Molecular Pathways: Targeting B7-H3 (CD276) for Human Cancer Immunotherapy. Clin Cancer Res. 2016 Jul 15;22(14):3425-3431. doi: 10.1158/1078-0432.CCR-15-2428. Epub 2016 May 20. PMID: 27208063; PMCID: PMC4947428.
3. Zhao B, Li H, Xia Y, Wang Y, Wang Y, Shi Y, Xing H, Qu T, Wang Y, Ma W. Immune checkpoint of B7-H3 in cancer: from immunology to clinical immunotherapy. J Hematol Oncol. 2022 Oct 25;15(1):153. doi: 10.1186/s13045-022-01364-7. PMID: 36284349; PMCID: PMC9597993.
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