KC-2422

293T-cyno-PD1-Cell-Line

24028

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Background of 293T-cyno-PD1-Cell-Line

PD-1 (programmed death receptor 1), also known as CD279 (cluster of differentiation 279), is an important immunosuppressive molecule. It regulates the immune system and promotes self-tolerance by down-regulating the immune system's response to human cells, as well as by suppressing T-cell inflammatory activity. This prevents autoimmune diseases, but it also prevents the immune system from killing cancer cells.

Specifications

Catalog Number	KC-2422
Cell Line Name	293T-cyno-PD1-Cell-Line
Host Cell Line	293T
Description	Stable HEK293T clone expressing exogenous cyno PD1 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% 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; 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

293T cyno-PD1 cell line was generated using a lentiviral vector expressing the cyno-PD1 sequence.

Characterization

Figure 1: Characterization of cyno-PD1 overexpression in the 293T cyno-PD1 stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 1μg/mL puromycin)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 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 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

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. doi: 10.3389/fcell.2020.00672. PMID: 32793604; PMCID: PMC7385189.
Arasanz H, Gato-Cañas M, Zuazo M, Ibañez-Vea M, Breckpot K, Kochan G, Escors D. PD1 signal transduction pathways in T cells. Oncotarget. 2017 Apr 19;8(31):51936-51945. doi: 10.18632/oncotarget.17232. PMID: 28881701; PMCID: PMC5584302.
Ngiow SF, Young A, Jacquelot N, Yamazaki T, Enot D, Zitvogel L, Smyth MJ. A Threshold Level of Intratumor CD8+ T-cell PD1 Expression Dictates Therapeutic Response to Anti-PD1. Cancer Res. 2015 Sep 15;75(18):3800-11. doi: 10.1158/0008-5472.CAN-15-1082. Epub 2015 Jul 24. PMID: 26208901. .

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.