KC-4447

293T human NRP1 Cell Line

46951

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Background of 293T human NRP1 Cell Line

NRP1, This gene encodes one of two neuropilins, which contain specific protein domains which allow them to participate in several different types of signaling pathways that control cell migration. Neuropilins contain a large N-terminal extracellular domain, made up of complement-binding, coagulation factor V/VIII, and meprin domains. These proteins also contains a short membrane-spanning domain and a small cytoplasmic domain. Neuropilins bind many ligands and various types of co-receptors; they affect cell survival, migration, and attraction. Some of the ligands and co-receptors bound by neuropilins are vascular endothelial growth factor (VEGF) and semaphorin family members. This protein has also been determined to act as a co-receptor for SARS-CoV-2 (which causes COVID-19) to infect host cells.

Specifications

Catalog Number	KC-4447
Cell Line Name	293T human NRP1 Cell Line
Host Cell Line	293T
Description	293T cell line stably expressing exogenous human 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 + 1 μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Fibroblastoid cells growing as monolayer
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-human-NRP1 cell line was generated using a lentiviral vector expressing the human NRP1 sequence.

Characterization

Figure1: Characterization of human NRP1 overexpression in 293T human NRP1 stable clones using quantitative real-time PCR.

Figure2: Characterization of 293T human NRP1 cell line stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 1 μg/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 9.0 mL 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

Ma Y, Zhang Z, Chen R, Shi R, Zeng P, Chen R, Leng Y, Chen AF. NRP1 regulates HMGB1 in vascular endothelial cells under high homocysteine condition. Am J Physiol Heart Circ Physiol. 2019 May 1;316(5):H1039-H1046. doi: 10.1152/ajpheart.00746.2018. Epub 2019 Feb 15. PMID: 30767669.
Battin C, De Sousa Linhares A, Paster W, Isenman DE, Wahrmann M, Leitner J, Zlabinger GJ, Steinberger P, Hofer J. Neuropilin-1 Acts as a Receptor for Complement Split Products. Front Immunol. 2019 Sep 13;10:2209. doi: 10.3389/fimmu.2019.02209. PMID: 31572401; PMCID: PMC6753332.
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.