KC-0234

293T-GITRL-Cell-Line

20067

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Background of 293T-GITRL-Cell-Line

GITRL, also known as TNF superfamily member 18,a type II transmembrane protein, is a ligand for glucocorticoidinduced TNFR-related protein (GITR). It has been shown to modulate T lymphocyte survival in peripheral tissues. This cytokine is also found to be expressed in endothelial cells and is thought to be important for the interaction between T lymphocytes and endothelial cells. Recent evidence suggests that the GITR/GITRL system participates in the development of inflammatory responses, including shock, either due to the early response of neutrophils and macrophages or together with autoimmune/allergic pathogenesis.

Specifications

Catalog Number	KC-0234
Cell Line Name	293T-GITRL-Cell-Line
Host Cell Line	Human HEK293T cell line
Description	Stable HEK293T cell line expressing exogenous human GITRL 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+0.5µg/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 human GITRL cell line was generated using a lentiviral vector expressing the human GITRL sequence.

Characterization

Figure: Characterization of GITRL overexpression in 293T stable clones using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS + 0.5μg/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

Krausz LT, Bianchini R, Ronchetti S, Fettucciari K, Nocentini G, Riccardi C. GITR-GITRL System, A Novel Player in Shock and Inflammation .The Scientific World JOURNAL, (2007) 7, 533ÿ566
Tang X, Tian J, Ma J, Wang J, Qi C, Rui K, Wang Y, Xu H, Lu L, Wang S. GITRL modulates the activities of p38 MAPK and STAT3 to promote Th17 cell differentiation in autoimmune arthritis. Oncotarget. 2016 Feb 23;7(8):8590- 600.
Kim JD, Choi BK, Bae JS, Lee UH, Han IS, Lee HW, Youn BS, Vinay DS, Kwon BS. Cloning and characterization of GITR ligand. Genes Immun. 2003 Dec;4(8):564-9.