KC-1001

293T-cyno-PDL1 Cell Line

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Background of 293T-cyno-PDL1 Cell Line

PD-L1 (Programmed Death-Ligand 1) , also known as CD274 or B7-H1, is an immune checkpoint protein expressed on antigen-presenting cells and tumor cells. It binds to PD-1 on T cells, suppressing T-cell activation and promoting immune evasion. Cynomolgus monkey PD-L1 (cyno-PDL1) shares high sequence homology with human PD-L1, making it a valuable preclinical model for evaluating anti-PD-1/PD-L1 immunotherapies. Overexpression of cyno-PDL1 in 293T cells enables studies of antibody binding, blockade efficacy, and cell-based functional assays for drug development targeting the PD-1/PD-L1 axis.

Specifications

Catalog Number	KC-1001
Cell Line Name	293T-cyno-PDL1 Cell Line
NCBI/UniProt Accession Number	XM_005581779.5
Clone Number	1-4#
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous cyno PDL1 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
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

The 293T-cyno-PDL1 cell line was generated using a lentiviral vector expressing the cyno PDL1 sequence.

Characterization

Figure 1: Characterization of cyno PDL1 overexpression in the 293T-cyno-PDL1 stable clone using FACS

Cell Resuscitation

Pre-warm complete culture medium (basal medium and 10% FBS) in a 37°C water bath.
Rapidly thaw the cryovial in a 37°C water bath for 1-2 minutes with gentle agitation.
Transfer the vial to a biosafety cabinet, and disinfect the exterior with 70% ethanol.
Aseptically transfer the cell suspension dropwise into a sterile centrifuge tube containing 9.0 mL of pre-warmed complete medium.
Centrifuge at approximately 125 × g for 5–7 minutes at room temperature, carefully aspirate the supernatant without disturbing the cell pellet.
Gently resuspend the pellet in an appropriate volume of complete medium and transfer the suspension into a T25 flask.
Incubate the flask in a 37°C in a humidified 5% CO₂ incubator.
Assess cell viability and morphology after 24 hours. If cells appear healthy, replace the medium with fresh medium supplemented with the appropriate selective antibiotic.
Subculture the cells at a ratio of 1:4-1:8 every 2-3 days upon reaching 80%–90% confluency.

Cell Freezing

Prepare the freezing medium (70% basal medium, 20% FBS and 10% DMSO) freshly before use.
Pre-chill the freezing medium on ice and label the cryovials accordingly.
Transfer the cell suspension to a sterile conical tube and perform a cell count to determine total viability and density.
Centrifuge the cells at 250×g for 5 minutes at room temperature; carefully aspirate the supernatant.
Gently resuspend the cell pellet in chilled freezing medium, ensuring a minimum cell density of 3×10⁶ cells/mL.
Aliquot 1 mL of the cell suspension into each pre-labeled cryovial.
Place the cryovials into a CoolCell® container and store at -80°C overnight for controlled-rate cooling.
Transfer the cryovials to the liquid nitrogen for long-term storage the following day.

References

1. Daassi, Dhouha et al. “The importance of exosomal PDL1 in tumour immune evasion.” Nature reviews. Immunology vol. 20,4 (2020): 209-215. doi:10.1038/s41577-019-0264-y
2. Zhang, Yun-Chao et al. “What role does PDL1 play in EMT changes in tumors and fibrosis?.” Frontiers in immunology vol. 14 1226038. 15 Aug. 2023, doi:10.3389/fimmu.2023.1226038
3. Wu, Jianheng, and Nannan Wang. “Current progress of anti‑PD‑1/PDL1 immunotherapy for glioblastoma (Review).” Molecular medicine reports vol. 30,6 (2024): 221. doi:10.3892/mmr.2024.13344