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	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% 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..