KC-5131

SHP77-DLL3-PDL1-KO Cell Line

55538

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Background of SHP77-DLL3-PDL1-KO Cell Line

PD-L1 is a 290 aa type I transmembrane protein encoded by the Cd274 gene on mouse chromosome 19 and human chromosome 9. Cd274 comprises seven exons, the first of which is noncoding and contains the 5′UTR. The next three exons contain the signal sequence, IgV-like domain, and IgC-like domains, respectively. The transmembrane domain and the intracellular domains are contained in the next two exons (exons 5 and 6). The last exon contains intracellular domain residues plus the 3′UTR. The intracellular domain of PD-L1 is short, only about 30 aa, and highly conserved in all reported species. There is no known function for the intracellular tail of PD-L1.There is one reported splice variant of PD-L1 in humans consisting of a sequence lacking the IgV-like domain encoded in exon 2. This mutant should not be able to bind PD-1, although the function of this splice variant has not yet been reported. No splice variants have been identified for mouse PD-L1.

Specifications

Catalog Number	KC-5131
Cell Line Name	SHP77-DLL3-PDL1-KO Cell Line
Clone Number	3B4
Host Cell Line	SHP77-DLL3-KO
Description	Stable SHP77-DLL3-KO clone with human PDL1 gene knockout
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	RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10% FBS
Selection Marker	NA
Morphology	rounded
Subculture	Split saturated culture 1:2-1:4 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

SHP77-DLL3-PDL1-KO cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of SHP77-DLL3-PDL1-KO cell line stable clone using PCR sequencing.

Figure 2: Characterization of SHP77-DLL3-PDL1-KO cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of SHP77-DLL3-PDL1-KO cell line stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (RPMI1640+10% FBS)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 1:2-1:4 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 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

Keir ME, Butte MJ, Freeman GJ, Sharpe AH. PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol. 2008;26:677-704. doi: 10.1146/annurev.immunol.26.021607.090331. PMID: 18173375; PMCID: PMC10637733.