KC-2122

B16/F10-mPDL1-KO-hPDL1-Low Cell Line

23434

Home » Cell Lines » B16/F10-mPDL1-KO-hPDL1-Low Cell Line

Background of B16/F10-mPDL1-KO-hPDL1-Low Cell Line

Programmed cell death 1 ligand 1 (PD-L1), encoded by the CD274 gene and best known as B7-H1, is a type I transmembrane ligand of the PD-1 inhibitory receptor. It is expressed on hematopoietic and many non-hematopoietic cells and is upregulated on a wide range of tumor cells. By engaging PD-1 on activated T cells, PD-L1 attenuates T-cell receptor signaling, suppresses cytotoxic T-cell activity and enables immune evasion in the tumor microenvironment. Elevated tumor PD-L1 is used as a companion diagnostic, and blocking antibodies such as atezolizumab, durvalumab and avelumab are established cancer immunotherapies that reinvigorate anti-tumor T-cell immunity.

Specifications

Catalog Number	KC-2122
Cell Line Name	B16/F10-mPDL1-KO-hPDL1-Low Cell Line
NCBI/UniProt Accession Number	NM_014143.4
Clone Number	4#
Host Cell Line	B16/F10-mPDL1-KO-1A4
Description	The B16/F10-mPDL1-KO-hPDL1-Low cell line stably overexpresses human CD274 (PD-L1) at low level with endogenous mouse PDL1 knocked out
Quantity	One vial of frozen cells (≥2×10⁶ cells)
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-like
Subculture	Split at a ratio of 1:4–1:6 every 2–3 days
Incubation	37°C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Estimated 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

The B16/F10-mPDL1-KO-hPDL1-Low cell line was generated using a lentiviral vector expressing the human CD274 sequence, with mouse PDL1 knocked out using CRISPR/Cas9 gene editing.

Characterization

Figure 1: Characterization of human PDL1 overexpression in the B16/F10-mPDL1-KO-hPDL1-Low 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 1:4–1:6 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 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

Keir ME, Butte MJ, Freeman GJ, Sharpe AH. PD-1 and its ligands in tolerance and immunity. Annual Review of Immunology. 2008.
Topalian SL, Drake CG, Pardoll DM. Immune checkpoint blockade: a common denominator approach to cancer therapy. Cancer Cell. 2015.
Sun C, Mezzadra R, Schumacher TN. Regulation and function of the PD-L1 checkpoint. Immunity. 2018.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.