KC-4412
293T-GARP-LTGFβ-Cell-Line
Background of 293T-GARP-LTGFβ-Cell-Line
GARP is a type I transmembrane cell surface docking receptor that mediates latent transforming growth factor - β (TGF - β), which is highly expressed 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.
Transforming growth factor-betas (TGF-betas) are multifunctional cytokines that exist in 3 isoforms in mammals. The TGF-betas are ubiquitously expressed and all isoforms are secreted as biologically inactive precursors called latent TGF-beta (L-TGF-beta).
Transforming growth factor-betas (TGF-betas) are multifunctional cytokines that exist in 3 isoforms in mammals. The TGF-betas are ubiquitously expressed and all isoforms are secreted as biologically inactive precursors called latent TGF-beta (L-TGF-beta).
Specifications
| Catalog Number | KC-4412 |
|---|---|
| Cell Line Name | 293T-GARP-LTGFβ-Cell-Line |
| Host Cell Line | 293T |
| Description | Stable 293T clone expressing exogenous GARP and LTGFβ 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-LTGFβ-cell-line was generated using a lentiviral vector expressing the GARP and LTGFβ sequence.
Characterization
Figure 1: Characterization of GARP-LTGFβ overexpression in the 293T-GARP-LTGFβ stable clone using FACS.
Figure 2: Characterization of GARP-LTGFβ in the 293T-GARP-LTGFβ stable clone using PCR 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.
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.
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.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.
2. 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.
3. Khalil N. Post translational activation of latent transforming growth factor beta (L-TGF-beta): clinical implications. Histol Histopathol. 2001 Apr;16(2):541-51. doi: 10.14670/HH-16.541. PMID: 11332710.
2. 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.
3. Khalil N. Post translational activation of latent transforming growth factor beta (L-TGF-beta): clinical implications. Histol Histopathol. 2001 Apr;16(2):541-51. doi: 10.14670/HH-16.541. PMID: 11332710.