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Chemokines belong to the cytokine family and function as chemotactic cytokines that regulate the migration and activation of phagocytes and lymphocytes. Chemokine receptors are G protein-coupled, seven-transmembrane cell-surface receptors characterized by high-affinity ligand binding. To date, at least 20 chemokine receptors have been identified, categorized into four subtypes: CXC chemokine receptors (CXCRs), CC chemokine receptors (CCRs), XCR, and CX3CR.

CC motif chemokine receptor 6 (CCR6) is a transmembrane G protein-coupled receptor. It exhibits the characteristic amino acid residues and sequence motifs of G protein-coupled chemokine receptors, including two potential N-linked glycosylation sites in the acidic N-terminal region and two additional glycosylation sites located on the first and third extracellular loops, respectively. Furthermore, the primary structure of CCR6 contains four highly conserved cysteine ​​residues (two forming a disulfide bridge and two remaining unpaired). Unlike other chemokine receptors, it binds exclusively to the ligand CCL20, forming the CCR6/CCL20 axis. CCR6 is expressed on various leukocyte subsets—including polymorphonuclear (PMN) cells, dendritic cells (DCs), T cells, and natural killer (NK) cells—with preferential expression on immature DCs and memory T cells. Recent studies have revealed that the CCR6/CCL20 axis is highly expressed in various human tumor cells and is closely associated with tumor invasion and metastasis.

The CHO-K1 Human CCR6 Cell Line model effectively simulates the in vivo CCR6 signal transduction process, the principle is illustrated in the figure below.

Figure 1. Schematic diagram of the CHO-K1 Human CCR6 Cell Line model

Figure 2. Recombinant CCR6 CHO-K1 stably expressing CCR6.

Figure 3. HTRF cAMP Assay with CCR6 CHO-K1(C3).

Cell Resuscitation
1)Rapidly thaw the frozen cells in a 37 °C water bath for approximately 60 seconds. Once thawed (which may take slightly less or more than 60 seconds), immediately transfer the cell suspension from the cryovial into a 15 mL centrifuge tube containing 10 mL of pre-warmed CHO-K1 Human CCR6 Cell Line complete culture medium.
2)Centrifuge cells at 1000 rpm for 5 min to remove medium, then resuspend cells in 5 mL of pre-warmed complete medium.
3)Transfer the cell suspension into a T25 culture flask and incubate at 37 °C with 5% CO₂.
4)After approximately 24–36 hours, replace the medium or passage the cells to remove non-adherent dead cells.

Subculturing procedure
1)When the cell density reaches the appropriate confluency for passaging, wash the cells with PBS, then add 1 mL trypsin to detach the cells. When more than 80% of the cells detach upon gently tapping the culture flask, add complete culture medium to terminate digestion. Gently pipette to obtain a single-cell suspension, transfer to a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes.

2)Discard supernatant after centrifugation. Resuspend cells in fresh medium to a single-cell suspension and transfer to a new culture flask for continued growth.

Cell Freezing
After trypsinization and centrifugation of cells from each T75 flask or 10 cm culture dish, discard the supernatant. Add 2 mL of cryopreservation medium (90% FBS + 10% DMSO), gently resuspend thoroughly, and aliquot into two cryovials. Immediately place the cryovials into a controlled-rate freezing container (e.g., Nalgene 5100-0001), fill with isopropanol to the indicated level, and store at −80 °C. After 24 hours, transfer the cryovials to liquid nitrogen for long-term storage.