KC-5631

CHOK1-rat-IL31RA-Flag Cell Line

59950

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Background of CHOK1-rat-IL31RA-Flag Cell Line

IL31RA (Interleukin 31 Receptor A) is a Protein Coding gene. The protein encoded by this gene belongs to the type I cytokine receptor family. This receptor, with homology to gp130, is expressed on monocytes, and is involved in IL-31 signaling via activation of STAT-3 and STAT-5. Type 2 inflammatory cytokines such as interleukin (IL)-4, IL-13, and IL-31 are strongly implicated in AD pathogenesis. Stimulation of IL-31 cognate receptors on C-fiber nerve endings is believed to activate neurons in the dorsal root ganglion (DRG), causing itch. The IL-31 receptor is a heterodimer of OSMRβ and IL31RA subunits, and OSMRβ can also bind oncostatin M (OSM), a pro-inflammatory cytokine released by monocytes/macrophages, dendritic cells, and T lymphocytes. IL-31 receptor α-chain (IL-31RA) is also expressed in peripheral nerves and epidermal keratinocytes, and the roles of IL-31 on pruritus and skin barrier have been investigated.

Specifications

Catalog Number	KC-5631
Cell Line Name	CHOK1-rat-IL31RA-Flag Cell Line
NCBI/UniProt Accession Number	NM_001257278.2
Clone Number	24#
Host Cell Line	CHOK1
Description	Stable CHOK1 clone expressing exogenous rat IL31RA Flag 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 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 24 hours
Mycoplasma Status	Negative

Cell Line Generation

CHOK1-rat-IL31RA-Flag cell line was generated using a lentiviral vector expressing the rat IL31RA Flag sequence.

Characterization

Figure 1: Characterization of endogenous Flag tag expression in CHOK1 cell using FACS.

Figure 2: Characterization of Flag tag overexpression in the CHOK1-rat-IL31RA-Flag stable clone using FACS.

Figure 3: Characterization of rat IL31RA in the CHOK1-rat-IL31RA-Flag stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 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 1-3 × 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. Datsi A, Steinhoff M, Ahmad F, Alam M, Buddenkotte J. Interleukin-31: The "itchy" cytokine in inflammation and therapy. Allergy. 2021 Oct;76(10):2982-2997. doi: 10.1111/all.14791. Epub 2021 Mar 16. PMID: 33629401.
2. Suehiro M, Numata T, Saito R, Yanagida N, Ishikawa C, Uchida K, Kawaguchi T, Yanase Y, Ishiuji Y, McGrath J, Tanaka A. Oncostatin M suppresses IL31RA expression in dorsal root ganglia and interleukin-31-induced itching. Front Immunol. 2023 Nov 16;14:1251031. doi: 10.3389/fimmu.2023.1251031. PMID: 38035099; PMCID: PMC10687395.
3. Nakashima C, Otsuka A, Kabashima K. Interleukin-31 and interleukin-31 receptor: New therapeutic targets for atopic dermatitis. Exp Dermatol. 2018 Apr;27(4):327-331. doi: 10.1111/exd.13533. PMID: 29524262.