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Figure 1. Recombinant CCR4 β-Arrestin/CHO-K1 stably expressing CCR4

Blue curve: CCR4 β-Arrestin/CHO-K1 cells stained with anti‑CCR4 antibody, mean fluorescence intensity (MFI) = 5.46E5. Red curve: Parental CHO-K1 cells as negative control, MFI = 852. The obvious peak shift verifies high stable membrane expression of human CCR4 receptor in this β-arrestin functional cell model.

Figure 2. Dose Response of Recombinant Human CCL22 in CCR4 β-Arrestin CHO(C12)

The β-arrestin recruitment signal (RLU) was measured upon CCL22 stimulation. EC50 = 32.4 ng/ml, Signal-to-background ratio (S/B) =7.3. The sigmoidal curve confirms the functional activity of CCR4 receptor in this β-arrestin assay cell model.

Figure 3. Inhibition of CCL22-induced beta-Arrestin Recruitment in CCR4 β-Arrestin CHO (C12)

Three CCR4 antagonists were tested. AZD2098, IC50 = 759.9 nM; C-021 dihydrochloride, IC50 = 17.9 nM; GSK2239633A, IC50 = 583.8 nM. The inhibition curves demonstrate that this cell model is suitable for screening and potency evaluation of CCR4 antagonists

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 CCR4 β-Arrestin 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.