KC-5685

293t-kdr-low-cell-line-kc-5685

60555

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Background of 293t-kdr-low-cell-line-kc-5685

Vascular endothelial growth factor (VEGF) is a major growth factor for endothelial cells, it is a Protein Coding gene. The vascular endothelial growth factor receptor 2 (VEGFR2) is a tyrosine kinase receptor regulating a variety of biological processes, including embryonic development, angiogenesis, tissue homeostasis and cancer. It functions as the main mediator of VEGF-induced endothelial proliferation, survival, migration, tubular morphogenesis and sprouting. The signalling and trafficking of this receptor are regulated by multiple factors, including Rab GTPase, P2Y purine nucleotide receptor, integrin alphaVbeta3, T-cell protein tyrosine phosphatase. Mutations of this gene are implicated in infantile capillary hemangiomas. Diseases associated with KDR include Hemangioma, Capillary Infantile and Squamous Cell Carcinoma, Head And Neck.

Specifications

Catalog Number	KC-5685
Cell Line Name	293t-kdr-low-cell-line-kc-5685
NCBI/UniProt Accession Number	NM_002253.4
Clone Number	10#
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous human KDR gene in low level
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-KDR-Low cell line was generated using a lentiviral vector expressing the human KDR sequence.

Characterization

Figure 1: Characterization of human KDR overexpression in the 293T-KDR-Low stable clone using FACS.

Figure 2: Characterization of human KDR in the 293T stable clone 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.Paradowska-Gorycka A, Stypinska B, Pawlik A, Malinowski D, Romanowska-Prochnicka K, Manczak M, Olesinska M. KDR (VEGFR2) Genetic Variants and Serum Levels in Patients with Rheumatoid Arthritis. Biomolecules. 2019 Aug 9;9(8):355. doi: 10.3390/biom9080355. PMID: 31405022; PMCID: PMC6727087.
2.Grillo E, Romani C, Ettorre VM, Santin AD, Mitola S. The VEGF/VEGFR2 system in ovarian cancer: From functional to pharmacological significance. Biochim Biophys Acta Rev Cancer. 2025 Sep;1880(4):189374. doi: 10.1016/j.bbcan.2025.189374. Epub 2025 Jun 16. PMID: 40516635.
3.Rezzola S, Di Somma M, Corsini M, Leali D, Ravelli C, Polli VAB, Grillo E, Presta M, Mitola S. VEGFR2 activation mediates the pro-angiogenic activity of BMP4. Angiogenesis. 2019 Nov;22(4):521-533. doi: 10.1007/s10456-019-09676-y. Epub 2019 Jul 30. PMID: 31363885.