KC-0255

293T-cyno-CD137-Cell-Line

20109

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Background of 293T-cyno-CD137-Cell-Line

CD137, also named 4-1BB ligand receptor and TNFRSF9, is a member of the tumor necrosis (TNF) receptor family and a famous co-stimulatory immune checkpoint molecule. CD137 is expressed on activated T cells, and nature killer cells, crosslinking of CD137 by agonistic antibody or its ligand enhance T cell proliferation, IL2 secretion and cytolytic activities.

Specifications

Catalog Number	KC-0255
Cell Line Name	293T-cyno-CD137-Cell-Line
Host Cell Line	Human HEK293T cell line
Description	HEK293T cell line stable expressing exogenous cynomolgus CD137 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 1:3-1:6 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 cyno CD137 cell line was generated using lentiviral vector expressing cynomolgus CD137 sequence.

Characterization

Figure: Characterization of CD137 overexpressing in 293T stable clones using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS + 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 1:3-1:6 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

Wolfl, M, J Kuball, W Y Ho, H Nguyen, T J Manley, M Bleakley, and P D Greenberg. 2007. ¡ùActivation-Induced Expression of CD137 Permits Detection, Isolation, and Expansion of the Full Repertoire of CD8+ T Cells Responding to Antigen Without Requiring Knowledge of Epitope Specificities.¡ì Blood 110 (1). American Society of Hematology: 201Ã¿10. doi:10.1182/blood-2006-11-056168.
Litjens, N H R, E A de Wit, C C Baan, and M G H Betjes. 2013. ¡ùActivation-Induced CD137 Is a Fast Assay for Identification and Multi-Parameter Flow Cytometric Analysis of Alloreactive T Cells.¡ì Clinical and Experimental Immunology 174 (1): 179Ã¿91. doi:10.1111/cei.12152.