KC-4465

293T-GARP-Cell-Line

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Background of 293T-GARP-Cell-Line

GARP, also known as LRRC32, is a type I transmembrane cell surface docking receptor that mediates the high expression of potential transforming growth factor beta (TGF - β) on regulatory T lymphocytes and platelets. It is a transmembrane protein highly expressed by activated Tregs and plays a key role in the activation of TGF - β, thereby promoting the immunosuppressive effect of Tregs. The overexpression of GARP in human cancers is associated with tolerance to TME and adverse clinical reactions to ICB, indicating that GARP blockade may improve cancer immunotherapy.

Specifications

Catalog Number	KC-4465
Cell Line Name	293T-GARP-Cell-Line
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous GARP 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

Cell Line Generation

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

Characterization

Figure 1: Characterization of GARP overexpression in the 293T-GARP stable clone using FACS.

Figure 2: Characterization of GARP in the 293T-GARP stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 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 1:4-1:8 every 2-3 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

Bellaye PS, Dias AM, Vrigneaud JM, Bouchard A, Moreau M, Petitot C, Bernhard C, Claron M, Froidurot L, Morgand V, Guillemin M, Monterrat M, Mirjolet C, Garrido C, Kohli E, Collin B. Targeted radionuclide therapy against GARP expressing T regulatory cells after tumour priming with external beam radiotherapy in a murine syngeneic model. Heliyon. 2024 Oct 18;10(20):e39543. doi: 10.1016/j.heliyon.2024.e39543. PMID: 39498075; PMCID: PMC11533616.
Li A, Chang Y, Song NJ, Wu X, Chung D, Riesenberg BP, Velegraki M, Giuliani GD, Das K, Okimoto T, Kwon H, Chakravarthy KB, Bolyard C, Wang Y, He K, Gatti-Mays M, Das J, Yang Y, Gewirth DT, Ma Q, Carbone D, Li Z. Selective targeting of GARP-LTGFβ axis in the tumor microenvironment augments PD-1 blockade via enhancing CD8+ T cell antitumor immunity. J Immunother Cancer. 2022 Sep;10(9):e005433. doi: 10.1136/jitc-2022-005433. PMID: 36096533; PMCID: PMC9472209.