KC-3284

293T-rat-TF Cell Line

34292

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Background of 293T-rat-TF Cell Line

Transferrin is a major glycoprotein in the blood that is responsible for transporting iron from liver tissue (where iron is primarily stored) to cells in other tissues.All growing cells have receptors on their surfaces for iron-binding transferrin, which binds to iron at neutral pH and then enters the cell through endocytosis. Inside the cell, in the acidic environment of the endosome, transferrin releases iron, but still binds to the membrane receptor and returns to the plasma membrane with the receptor. When the extracellular environment becomes neutral, transferrin breaks away from the receptor, binds freely to iron, and begins the cycle again. In effect, transferrin shuttles between the extracellular fluid and the endosome, avoiding the lysosomes and rapidly delivering the iron needed for cell growth.

Specifications

Catalog Number	KC-3284
Cell Line Name	293T-rat-TF Cell Line
Host Cell Line	293T
Description	Stable HEK293T clone expressing exogenous rat-TF 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 + 1μg/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
In Vivo Validation	NA

Cell Line Generation

293T-rat-TF cell line was generated using a lentiviral vector expressing the rat-TF sequence.

Characterization

Figure 1: Characterization of rat-TF in the 293T rat-TF stable clone using qPCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 1μg/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% DMEM + 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. Kawabata H. Transferrin and transferrin receptors update. Free Radic Biol Med. 2019 Mar;133:46-54. doi: 10.1016/j.freeradbiomed.2018.06.037. Epub 2018 Jun 30. PMID: 29969719. 2.Bentsen M, Heger V, Schultheis H, Kuenne C, Looso M. TF-COMB - Discovering grammar of transcription factor binding sites. Comput Struct Biotechnol J. 2022 Jul 21;20:4040-4051. doi: 10.1016/j.csbj.2022.07.025. PMID: 35983231; PMCID: PMC9358416. 3.Ramsey J, Mukhopadhyay S. Disentangling the Frames, the State of Research on the Alphavirus 6K and TF Proteins. Viruses. 2017 Aug 18;9(8):228. doi: 10.3390/v9080228. PMID: 28820485; PMCID: PMC5580485.