KC-0219

293T-PDL2-Cell-Line

20037

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Background of 293T-PDL2-Cell-Line

Human programed cell death ligand 2 (PD-L2), also named B7-DC, is membrane of the B7 immune regulating protein. PD-L2 is expressed on dendritic cells, activated T cells and memory B cells. PD-L2 cannot only provide the costimulatory signal for T-cell proliferation and IFNG production in a PDCD1-independent manner, but also inhibits T-cell proliferation by blocking cell cycle progression and cytokine production through interaction with PDCD1. Recent studies showed that PD-L2 is a promising drug target for cancer immune therapy.

Specifications

Catalog Number	KC-0219
Cell Line Name	293T-PDL2-Cell-Line
Host Cell Line	Human HEK293T cell line
Description	HEK293T cell line stable expressing exogenous human PD-L2 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 Human PD-L2 Cell Line was generated using plasmid with human PDCD1LG2 gene sequence.

Characterization

Figure: Characterization of PD-L2 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

Sunshine, J. & Taube, J. M. PD-1/PD-L1 inhibitors. Current Opinion in Pharmacology 23, 32ÿ38 (2015).
Boussiotis, V. A. Molecular and Biochemical Aspects of the PD-1 Checkpoint Pathway. N Engl J Med 375, 1767ÿ1778 (2016).
Topalian, S. L. et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 366, 2443ÿ2454 (2012).