KC-5781

MDA-MB-468-B7H4-KO Cell Line

59042

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Background of MDA-MB-468-B7H4-KO Cell Line

The coinhibitory molecule B7-H4, an important member of the B7 family, is abnormally expressed in tumors, inflammation and autoimmune diseases. B7-H4 negatively regulates T cell immune response and promotes immune escape by inhibiting the proliferation, cytokine secretion, and cell cycle of T cells. Moreover, B7-H4 plays an extremely important role in tumorigenesis and tumor development including cell proliferation, invasion, metastasis, anti-apoptosis, etc. In addition, B7-H4 has the other biological functions, such as protection against type 1 diabetes (T1D) and islet cell transplantation. Therefore, B7-H4 has been identified as a novel marker or a therapeutic target for the treatment of tumors, inflammation, autoimmune diseases, and organ transplantation.

Specifications

Catalog Number	KC-5781
Cell Line Name	MDA-MB-468-B7H4-KO Cell Line
Clone Number	1A2
Host Cell Line	MDA-MB-468
Description	Stable MDA-MB-468 clone with human B7H4 gene knockout, No.1A2
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% DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10% FBS
Selection Marker	NA
Morphology	Epithelial
Subculture	Split saturated culture 1:2-1:3 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 30-40 hours
Mycoplasma Status	Negative

Cell Line Generation

MDA-MB-468-B7H4-KO cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of MDA-MB-468-B7H4-KO cell line stable clone using PCR sequencing.

Figure 2: Characterization of MDA-MB-468-B7H4-KO cell line stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS) 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 1:2-1:3 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 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

Wang JY, Wang WP. B7-H4, a promising target for immunotherapy. Cell Immunol.2020 Jan;347:104008. doi: 10.1016/j.cellimm.2019.104008. Epub 2019 Nov 4. PMID:31733822.