KC-2432

293T-mouse-CD25-High-Cell-Line

24048

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Background of 293T-mouse-CD25-High-Cell-Line

CD25 (α chain of the high-affinity IL-2 receptor) deficiency is an autosomal recessive disorder due to mutations in the IL2RA gene. T regulatory cells constitutively express CD25 and respond to IL-2 generated by T cells during an immune response for their immunoregulatory functions. CD25 deficiency results in a syndrome similar to IPEX with severe enteropathy, diabetes mellitus, autoimmune hemolytic anemia, eczema, and lymphoproliferation. Importantly, patients with CD25 deficiency exhibit several unique features not seen in IPEX, namely chronic herpetic viral infections and an increased susceptibility to infections.

Specifications

Catalog Number	KC-2432
Cell Line Name	293T-mouse-CD25-High-Cell-Line
Host Cell Line	Human HEK293T cell line
Description	Stable 293T cell line expressing exogenous mouse CD25 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 1:3-1:6 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

293T-mouse-CD25-high cell line was generated using a lentiviral vector expressing the mouse CD25 sequence.

Characterization

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS + 1μg/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 1:3-1:6 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

James W. Verbsky, John R. Routes, Infections, Immune Disorders, and Autoinflammatory Diseases. Nelson Pediatric Symptom-Based Diagnosis, 2018.