KC-1840

293T-NFAT-Luc2-KDR Cell Line

22903

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Background of 293T-NFAT-Luc2-KDR Cell Line

KDR, also named as VEGFR2 or CD309, is a type IV receptor tyrosine kinase, and almost exclusively expressed on the endothelial cells, and play an essential role in the regulation of angiogenesis, vascular development, vascular permeability, and embryonic hematopoiesis, and promotes proliferation, survival, migration and differentiation of endothelial cells.

Specifications

Catalog Number	KC-1840
Cell Line Name	293T-NFAT-Luc2-KDR Cell Line
Host Cell Line	293T-NFAT-Luc2
Description	Stable HEK293T cell line expressing exogenous luciferase gene under the control of NFAT responsive element as well as exogenous KDR protein.
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 +150μg/ml Hygromycin B+1μg/ml Puromycin
Selection Marker	Puromycin, Hygromycin B
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
In Vivo Validation	NA

Cell Line Generation

293T-NFAT-Luc2-KDR cell line was generated using a lentiviral vector expressing the human KDR sequence.

Characterization

Figure 1. Characterization of KDR overexpression in 293T stable clones using FACS.

Figure 2. Recombinant Human VEGF165 Protein activates NFAT-Luc2 reporter in HEK293T human embryonic kidney cells transfected with human KDR. The EC50 for this effect is ~3 ng/mL.

Figure 3. Recombinant Mouse VEGF164 Protein activates NFAT-Luc2 reporter in HEK293T human embryonic kidney cells transfected with human KDR. The EC50 for this effect is ~1.8 ng/mL.

Figure 4. Recombinant Human FLT-1 Protein blocks the binding of VEGF165 to KDR on 293T-NFAT-KDR-Luc2 cells. The IC50 for this effect is ~60 ng/mL.

Figure 5. Recombinant Human FLT-1 Protein blocks the binding of VEGF164 to KDR on 293T-NFAT-KDR-Luc2 cells. The IC50 for this effect is ~50 ng/mL.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS, 150μg/mL Hygromycin 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 1:4-1:5 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

1.Holmes K, Roberts OL, Thomas AM, Cross MJ (October 2007). Vascular endothelial growth factor receptor-2: structure, function, intracellular signalling and therapeutic inhibition. Cellular Signalling. 19 (10): 2003–12.
2.Petrova TV, Makinen T, Alitalo K (November 1999). Signaling via vascular endothelial growth factor receptors. Experimental Cell Research. 253 (1): 117–30.
3.Sato Y, Kanno S, Oda N, Abe M, Ito M, Shitara K, Shibuya M (May 2000). Properties of two VEGF receptors, Flt-1 and KDR, in signal transduction. Annals of the New York Academy of Sciences. 902 (1): 201–5, discussion 205–7.