KC-2023

MC38-CD228-Cell-Line

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Background of MC38-CD228-Cell-Line

CD228,a mammalian iron-transport protein and a soluble form of melanotransferrin Melanotransferrin (CD228), firstly reported as a melanoma-associated antigen, is a membrane-bound glycoprotein of an iron-binding transferrin homolog. CD228 was found to be expressed significantly higher in human bone marrow-derived mesenchymal stem cells (hBM-MSC) than in human embryonic fibroblasts (FB) by RT-PCR, western blotting and flow cytometry.

Specifications

Catalog Number	KC-2023
Cell Line Name	MC38-CD228-Cell-Line
Host Cell Line	MC38
Description	Stable MC38 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% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 5μg/ml Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:10 every 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

MC38 CD228 Cell Line was generated using a lentiviral vector expressing the CD228 sequence.

Characterization

Figure 1: Characterization of CD228 overexpression in the MC38 CD228 stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 5μg/mL Puromycin)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 1:10 every 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

Dubon M, Lee S, Park JH, Lee JY, Kang D. The Role of Melanotransferrin (CD228) in the regulation of the differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells (hBM-MSC). Int J Med Sci. 2021 Feb 4;18(7):1580-1591. 2.Singh CSB, Eyford BA, Abraham T, Munro L, Choi KB, Okon M, Vitalis TZ, Gabathuler R, Lu CJ, Pfeifer CG, Tian MM, Jefferies WA. Discovery of a Highly Conserved Peptide in the Iron Transporter Melanotransferrin that Traverses an Intact Blood Brain Barrier and Localizes in Neural Cells. Front Neurosci. 2021 Jun 2;15:596976.