KC-1229

293T-CD25-Cell-Line

21753

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Background of 293T-CD25-Cell-Line

Glucocorticoid-induced TNFR-related protein (GITR), also named Tumor necrosis factor receptor superfamily member 18 (TNFRSF18) or activation-inducible TNFR family receptor (AITR), is a cell membrane protein receptor acting as a co-stimulatory immune checkpoint molecule on T cell activation, and has proved to play a critical role in inhibiting the suppressive activity of T-regulatory cells and regulating the TCR meditated T cell death.

Specifications

Catalog Number	KC-1229
Cell Line Name	293T-CD25-Cell-Line
NCBI/UniProt Accession Number	NM_000417.3
Host Cell Line	Human HEK293T cell line
Description	Stable HEK293T clone expressing exogenous human CD25
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 CD25 cell line was generated using lentiviral vector expressing human CD25 sequence.

Characterization

Figure: Characterization of CD25 overexpressing in 293T stable clones using FACS.

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. Nocentini, G., Ronchetti, S., Cuzzocrea, S. & Riccardi, C. GITR/GITRL: More than an effector T cell co-stimulatory system. Eur. J. Immunol. 37, 1165–1169 (2007).
2. Esparza, E. M. & Arch, R. H. TRAF4 functions as an intermediate of GITR-induced NF-kB activation. CMLS, Cell. Mol. Life Sci. 61, 3087–3092 (2004).
3. Wyzgol, A. et al. Trimer Stabilization, Oligomerization, and Antibody-Mediated Cell Surface Immobilization Improve the Activity of Soluble Trimers of CD27L, CD40L, 41BBL, and Glucocorticoid-Induced TNF Receptor Ligand. The Journal of Immunology 183, 1851–1861 (2009).