KC-5530

293T-mouse-NRP1 Cell Line

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Background of 293T-mouse-NRP1 Cell Line

Neuropilin 1 (NRP1) is a transmembrane glycoprotein that acts as a co-receptor for a number of extracellular ligands including class III/IV semaphorins, certain isoforms of vascular endothelial growth factor and transforming growth factor beta. The transmembrane receptor neuropilin-1 (NRP-1) is highly expressed by mesangial cells and its genetic deletion results in proteinuric disease and glomerulosclerosis. In animal experiments, our results demonstrate that NRP1 is an entry receptor for reovirus and uncover mechanisms by which NRPs promote viral entry and pathogenesis. Diseases associated with NRP1 include Covid-19 and Age Related Macular Degeneration.

Specifications

Catalog Number	KC-5530
Cell Line Name	293T-mouse-NRP1 Cell Line
Clone Number	5-5#
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous mouse NRP1 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

Cell Line Generation

293T-mouse-NRP1 cell line was generated using a lentiviral vector expressing the mouse NRP1 sequence.

Characterization

Figure 1: Characterization of mouse NRP1 overexpression in the 293T mouse NRP1 stable clone using FACS(Kyinno, Cat#KB-1596).

Figure 2: Characterization of mouse NRP1 in the 293T stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 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 1:4-1:8 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. Sharma, S., Ehrlich, M., Zhang, M. et al. NRP1 interacts with endoglin and VEGFR2 to modulate VEGF signaling and endothelial cell sprouting. Commun Biol 7, 112 (2024).
2. Chaudhary B, Khaled YS, Ammori BJ, Elkord E. Neuropilin 1: function and therapeutic potential in cancer. Cancer Immunol Immunother. 2014 Feb;63(2):81-99. doi: 10.1007/s00262-013-1500-0. Epub 2013 Nov 22. PMID: 24263240; PMCID: PMC11028473.
3. Torres-Salido MT, Sanchis M, Solé C, Moliné T, Vidal M, Vidal X, Solà A, Hotter G, Ordi-Ros J, Cortés-Hernández J. Urinary Neuropilin-1: A Predictive Biomarker for Renal Outcome in Lupus Nephritis. Int J Mol Sci. 2019 Sep 17;20(18):4601. doi: 10.3390/ijms20184601. PMID: 31533337; PMCID: PMC6769814.