KC-0213

293T-ICOSLG Cell Line

20025

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Background of 293T-ICOSLG Cell Line

Inducible T-cell costimulator (ICOS) belongs to the CD28 family of cosignaling molecules and binds to ICOS ligand (ICOSL), it also known as CD275. ICOSL/ICOS are costimulatory molecules pertaining to immune checkpoints; their binding transduces signals having anti-tumor activity. Autosomal recessive mutations in Inducible T Cell Costimulator Ligand (ICOSLG) result in a combined immunodeficiency syndrome of humans, saliently marked by recurrent respiratory tract infections and significant disease with DNA-based viruses at epithelial barriers, including human papillomavirus (HPV). Diseases associated with ICOSLG include Immunodeficiency 119 and Dyskeratosis Congenita, Autosomal Dominant 6.

Specifications

Catalog Number	KC-0213
Cell Line Name	293T-ICOSLG Cell Line
Clone Number	N/A
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous human ICOSLG 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-ICOSLG cell line was generated using a lentiviral vector expressing the human ICOSLG sequence.

Characterization

Figure 1: Characterization of human ICOSLG overexpression in the 293T stable clone 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. Roussel L, Vinh DC. ICOSL in host defense at epithelial barriers: lessons from ICOSLG deficiency. Curr Opin Immunol. 2021 Oct;72:21-26. doi: 10.1016/j.coi.2021.03.001. Epub 2021 Mar 21. PMID: 33756276.
2. Dong M, Chang J, Lebel MÈ, Gervais N, Fournier M, Mallet Gauthier È, Suh WK, Melichar HJ. The ICOS-ICOSL pathway tunes thymic selection. Immunol Cell Biol. 2022 Mar;100(3):205-217. doi: 10.1111/imcb.12520. Epub 2022 Jan 23. PMID: 34962663; PMCID: PMC9304562.
3. Raineri D, Dianzani C, Cappellano G, Maione F, Baldanzi G, Iacobucci I, Clemente N, Baldone G, Boggio E, Gigliotti CL, Boldorini R, Rojo JM, Monti M, Birolo L, Dianzani U, Chiocchetti A. Osteopontin binds ICOSL promoting tumor metastasis. Commun Biol. 2020 Oct 26;3(1):615. doi: 10.1038/s42003-020-01333-1. PMID: 33106594; PMCID: PMC7588454.