KC-0352

293T-CCR6 Cell Line

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Background of 293T-CCR6 Cell Line

This gene encodes a member of the beta chemokine receptor family, which is predicted to be a seven transmembrane protein similar to G protein-coupled receptors. The gene is preferentially expressed by immature dendritic cells and memory T cells. The ligand of this receptor is macrophage inflammatory protein 3 alpha (MIP-3 alpha). This receptor has been shown to be important for B-lineage maturation and antigen-driven B-cell differentiation, and it may regulate the migration and recruitment of dentritic and T cells during inflammatory and immunological responses. Alternatively spliced transcript variants that encode the same protein have been described for this gene.

Specifications

Catalog Number	KC-0352
Cell Line Name	293T-CCR6 Cell Line
NCBI/UniProt Accession Number	NM_004367.6
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous CCR6 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 1:4-1:5 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
In Vivo Validation	NA

Cell Line Generation

293T-CCR6 cell line was generated using a lentiviral vector expressing the CCR6 sequence.

Characterization

Figure 1: Characterization of CCR6 overexpression in the 293T-CCR6 stable clone using FACS.

Figure 2: Characterization of CCR6 overexpressing in 293T-CCR6 stable clone using PCR sequencing.

Cell Resuscitation

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

Kulkarni N, Meitei HT, Sonar SA, Sharma PK, Mujeeb VR, Srivastava S, Boppana R, Lal G. CCR6 signaling inhibits suppressor function of induced-Treg during gut inflammation. J Autoimmun. 2018 Mar;88:121-130. doi: 10.1016/j.jaut.2017.10.013. Epub 2017 Nov 7. PMID: 29126851.
Zhang XG, Song BT, Liu FJ, Sun D, Wang KX, Qu H. CCR6 overexpression predicted advanced biological behaviors and poor prognosis in patients with gastric cancer. Clin Transl Oncol. 2016 Jul;18(7):700-7. doi: 10.1007/s12094-015-1420-x. Epub 2015 Oct 21. PMID: 26489425.