KC-2544

CHOK1-CD228 Cell Line

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Background of CHOK1-CD228 Cell Line

CD228, also known as MELTF,is a cell-surface glycoprotein initially found on melanoma cells,and it belongs to the transferrin superfamily.MELTF is related to transferrin in tertiary structure and has the physiological role of iron cellular uptake.In malignancy,it has been reported that MELTF is significantly overexpressed in melanoma and associated with increased proliferation of melanoma cells.Additionally, recent reports have confirmed MELTF as a potential serological marker for colorectal cancer.

Specifications

Catalog Number	KC-2544
Cell Line Name	CHOK1-CD228 Cell Line
Clone Number	2#
Host Cell Line	CHOK1
Description	Stable CHOK1 cell line expressing exogenous CD228 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 + 10μg/ml Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:5 every 2-3 days; seed out at about 1-2 × 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

CHOK1-CD228 Cell Line was generated using a lentiviral vector expressing the CD228 sequence.

Characterization

Figure 1: Characterization of CD228 overexpression in the CHO-K1 CD228 stable clone using FACS.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 + 10% FBS + 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 1-2 × 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. Zhang L,Shi L.The E2F1/MELTF axis fosters the progression of lung adenocarcinoma by regulating the Notch signaling pathway.Mutat Res.2023 Jul-Dec;827:111837.
2. Sawaki K, Kanda M, Umeda S,et al.Level of Melanotransferrin in Tissue and Sera Serves as a Prognostic Marker of Gastric Cancer. Anticancer Res.2019 Nov;39(11):6125-6133.