KC-4686

CHOK1-CD228-short Cell Line

59058

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Background of CHOK1-CD228-short Cell Line

The protein encoded by CD228 is a cell-surface glycoprotein found on melanoma cells. The protein shares sequence similarity and iron-binding properties with members of the transferrin superfamily. The importance of the iron binding function has not yet been identified. This gene resides in the same region of chromosome 3 as members of the transferrin superfamily. Alternative splicing results in two transcript variants.

Specifications

Catalog Number	KC-4686
Cell Line Name	CHOK1-CD228-short Cell Line
NCBI/UniProt Accession Number	NP_005920.2
Clone Number	1-2#
Host Cell Line	CHOK1
Description	CHOK1 cell line stably expressing exogenous CD228 short 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	Epithelial
Subculture	Split saturated culture 1:4-1:8 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 hours
Mycoplasma Status	Negative

Cell Line Generation

CHOK1-CD228-short cell line was generated using a lentiviral vector expressing the CD228 short sequence.

Characterization

Figure1: Characterization of CD228-short overexpressing in CHOK1 stable clones using FACS.

Figure2: Characterization of CHOK1-CD228-short cell line stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS + 10 μg/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 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 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

Rothenberger S, Food MR, Gabathuler R, Kennard ML, Yamada T, Yasuhara O, McGeer PL, Jefferies WA. Coincident expression and distribution of melanotransferrin and transferrin receptor in human brain capillary endothelium. Brain Res. 1996 Mar 11;712(1):117-21. doi: 10.1016/0006-8993(96)88505-2. PMID: 8705293.
Shin J, Kim HJ, Kim G, Song M, Woo SJ, Lee ST, Kim H, Lee C. Discovery of melanotransferrin as a serological marker of colorectal cancer by secretome analysis and quantitative proteomics. J Proteome Res. 2014 Nov 7;13(11):4919-31. doi: 10.1021/pr500790f. Epub 2014 Sep 23. PMID: 25216327.
Suryo Rahmanto Y, Dunn LL, Richardson DR. The melanoma tumor antigen, melanotransferrin (p97): a 25-year hallmark--from iron metabolism to tumorigenesis. Oncogene. 2007 Sep 13;26(42):6113-24. doi: 10.1038/sj.onc.1210442. Epub 2007 Apr 23. PMID: 17452986.