KC-3185

CHOK1-TFRC-Cell-Line

34192

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Background of CHOK1-TFRC-Cell-Line

TFRC, also known as CD71, is a transmembrane glycoprotein that encodes a cell surface receptor. TfR1 is required for iron import from transferrin into cells by endocytosis. TFRC is a potential new target in cases of human leukomia & lymphoma. TFRC has been shown to interact with GABARAP and HFE.

Specifications

Catalog Number	KC-3185
Cell Line Name	CHOK1-TFRC-Cell-Line
Host Cell Line	CHOK1
Description	Stable CHOK1 clone expressing exogenous TFRC 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-like
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
In Vivo Validation	NA

Cell Line Generation

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

Characterization

Figure 1: Characterization of TFRC overexpression in the CHOK1-TFRC stable clone using FACS.

Figure 2: Characterization of TFRC in the CHOK1-TFRC stable clone using PCR sequencing.

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 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% 1640 + 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.Antineoplastic efficacy profiles of avapritinib and nintedanib in KIT D816V+ systemic mastocytosis: a preclinical study. Degenfeld-Schonburg, Gamperl, Stefanzl et al. Am J Cancer Res (2023) 13 (2), 355-378.
2.Aisen P (November 2004). Transferrin receptor 1. The International Journal of Biochemistry & Cell Biology. 36 (11): 2137–43. doi:10.1016/j.biocel.2004.02.007. PMID 15313461.
3.Moos T (November 2002). Brain iron homeostasis. Danish Medical Bulletin. 49 (4): 279–301. PMID 12553165.
4.Speeckaert MM, Speeckaert R, Delanghe JR (December 2010). Biological and clinical aspects of soluble transferrin receptor. Critical Reviews in Clinical Laboratory Sciences. 47 (5–6):213–28. doi:10.3109/10408363.2010.550461. PMID 21391831. S2CID 25425279.