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	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; 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. .