KC-4820

CHOK1-NPR3 Cell Line

57608

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Background of CHOK1-NPR3 Cell Line

NPR3 encodes one of three natriuretic peptide receptors. Natriutetic peptides are small peptides which regulate blood volume and pressure, pulmonary hypertension, and cardiac function as well as some metabolic and growth processes. The product of this gene encodes a natriuretic peptide receptor responsible for clearing circulating and extracellular natriuretic peptides through endocytosis of the receptor.

Specifications

Catalog Number	KC-4820
Cell Line Name	CHOK1-NPR3 Cell Line
Clone Number	4-5#
Host Cell Line	CHOK1
Description	Stable CHOK1 cell line expressing exogenous human NPR3 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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS + 10μg/ml Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-2 × 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

CHOK1 human NPR3 Cell Line was generated using a lentiviral vector expressing the human NPR3 sequence.

Characterization

Figure 1: Characterization of human NPR3 overexpression in the CHO-K1 human NPR3 stable clone using PCR sequence

Figure 2: Characterization of human NPR3 overexpression in the CHO-K1 human NPR3 stable clone using FACS.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 + 10% FBS + 10μg/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 1-2 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 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. Li S, Guo R, Peng Z, Quan B, Hu Y, Wang Y, Wang Y. NPR3, transcriptionally regulated by POU2F1, inhibits osteosarcoma cell growth through blocking the PI3K/AKT pathway. Cell Signal. 2021 Oct;86:110074.
2. Roos J, Dahlhaus M, Funcke JB, Kustermann M, Strauss G, Halbgebauer D, Boldrin E, Holzmann K, Möller P, Trojanowski BM, Baumann B, Debatin KM, Wabitsch M, Fischer-Posovszky P. miR-146a regulates insulin sensitivity via NPR3. Cell Mol Life Sci. 2021 Mar;78(6):2987-3003.