KC-3326

TOV112D-PDL1 Cell Line

26953

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Background of TOV112D-PDL1 Cell Line

PD-L1, also called human programmed cell death ligand 1, is a transmembrane protein that plays a significant rolein suppressing the immune system during particular events such as pregnancy, tissue allografts, autoimmune disease, virus infection, and cancer. PD-L1 binds to its receptor PD-1 on activated T cells, B cells, and myeloid cells to modulate activation or inhibition. Upregulation of PD-L1 can allow the cancer cell to evade the host immune system.

Specifications

Catalog Number	KC-3326
Cell Line Name	TOV112D-PDL1 Cell Line
Host Cell Line	TOV112D
Description	Stable TOV112D clone expressing exogenous PDL1 gene
Quantity	One vial 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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS + 1μg/ml Puromycin
Selection Marker	Puromycin
Morphology	Fibroblastoid cells growing as a monolayer
Subculture	Split saturated culture 1:4-1:5 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
In Vivo Validation	NA

Cell Line Generation

TOV112D PDL1 cell line was generated using a lentiviral vector expressing the PDL1 sequence.

Characterization

Figure 1: Characterization of PDL1 overexpression in TOV112D stable clones using FACS.

Figure 2: Characterization of PDL1 and its mutants overexpressing in TOV112D stable clones using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 1μg/mL puromycin)in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO₂ incubator.
8. Split saturated culture 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage.

References

1. Ning H, Chiu SH, Xu X, Ma Y, Chen JL, Yang G. The Immunosuppressive Roles of PD-L1 during Influenza A Virus Infection. Int J Mol Sci. 2023 May 11;24(10):8586.
2. Lei Q, Wang D, Sun K, Wang L, Zhang Y. Resistance Mechanisms of Anti-PD1/PDL1 Therapy in Solid Tumors. Front Cell Dev Biol. 2020 Jul 21;8:672.
3. Wang X, Teng F, Kong L, Yu J. PD-L1 expression in human cancers and its association with clinical outcomes. Onco Targets Ther. 2016 Aug 12;9:5023-39..

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.