KC-5922

293T-rabbit-PD1-Flag Cell Line

61096

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Background of 293T-rabbit-PD1-Flag 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-5922
Cell Line Name	293T-rabbit-PD1-Flag Cell Line
NCBI/UniProt Accession Number	Q66HP6
Clone Number	10#
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous rabbit PD1 Flag 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 1:4-1:5 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

Cell Line Generation

293T-rabbit-PD1-Flag cell line was generated using lentiviral vector expressing rabbit PD1 Flag sequence.

Characterization

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

Figure2: Characterization of endogenous rabbit PD1 Flag expression in 293T using FACS.

Figure 3: Characterization of rabbit PD1 Flagexpression in 293T-rabbit-PD1-Flag stable clones using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM + 10% FBS + 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% DMEM + 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.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.
2.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.