KC-6340

CHOK1-CLEC4C-Luc2 Cell Line

61317

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Background of CHOK1-CLEC4C-Luc2 Cell Line

The type II C-type lectin CLEC4C is a transmembrane protein selectively expressed on plasmacytoid dendritic cells (PDCs). This gene encodes a member of the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily. Members of this family share a common protein fold and have diverse functions, such as cell adhesion, cell-cell signalling, glycoprotein turnover, and roles in inflammation and immune response. Two transcript variants encoding distinct isoforms have been identified for this gene. CLEC4C is a key regulator of the functions of pDCs, and its research value spans multiple cutting-edge fields such as basic immunology, tumor diagnosis, and the development of new drugs for autoimmune diseases.

Specifications

Catalog Number	KC-6340
Cell Line Name	CHOK1-CLEC4C-Luc2 Cell Line
NCBI/UniProt Accession Number	NM_001371390.1
Clone Number	1#
Host Cell Line	CHOK1
Description	Stable CHOK1 clone expressing exogenous human CLEC4C and luciferase 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 +750μg/mL hygromycin B
Selection Marker	Puromycin \| Hygromycin B
Morphology	Epithelial
Incubation	37 °C with 5% CO₂
Storage	Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative

Cell Line Generation

CHOK1-CLEC4C-Luc2 cell line was generated using a lentiviral vector expressing the human CLEC4C and luciferase sequence.

Characterization

Figure 1: Characterization of human CLEC4C overexpression in the CHOK1-CLEC4C-Luc2 stable clone using FACS.

Figure 2: Characterization of the CHOK1-CLEC4C-Luc2 cell line stable clone using Bright-Lite Luciferase Assay System in the conditions of different cell number.

Figure 3: Characterization of human CLEC4C in the CHOK1 stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 + 10% FBS + 10μg/mL Puromycin +750μg/mL hygromycin B)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. Murray L, Xi Y, Upham JW. CLEC4C gene expression can be used to quantify circulating plasmacytoid dendritic cells. J Immunol Methods. 2019 Jan;464:126-130. doi: 10.1016/j.jim.2018.11.001. Epub 2018 Nov 3. PMID: 30395816.
2. Riboldi E, Daniele R, Parola C, Inforzato A, Arnold PL, Bosisio D, Fremont DH, Bastone A, Colonna M, Sozzani S. Human C-type lectin domain family 4, member C (CLEC4C/BDCA-2/CD303) is a receptor for asialo-galactosyl-oligosaccharides. J Biol Chem. 2011 Oct 14;286(41):35329-35333. doi: 10.1074/jbc.C111.290494. Epub 2011 Aug 31. PMID: 21880719; PMCID: PMC3195614.
3. Werth VP, Furie RA, Romero-Diaz J, Navarra S, Kalunian K, van Vollenhoven RF, Nyberg F, Kaffenberger BH, Sheikh SZ, Radunovic G, Huang X, Clark G, Carroll H, Naik H, Gaudreault F, Meyers A, Barbey C, Musselli C, Franchimont N; LILAC Trial Investigators. Trial of Anti-BDCA2 Antibody Litifilimab for Cutaneous Lupus Erythematosus. N Engl J Med. 2022 Jul 28;387(4):321-331. doi: 10.1056/NEJMoa2118024. PMID: 35939578.
4. Greer LF 3rd, Szalay AA. Imaging of light emission from the expression of luciferases in living cells and organisms: a review. Luminescence. 2002 Jan-Feb;17(1):43-74. doi: 10.1002/bio.676. PMID: 11816060.