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Figure 1. Recombinant CCR8 β-Arrestin/CHO-K1 stably expressing CCR8

Cells were stained with anti-CCR8 antibody. The blue curve represents CCR8 β-Arrestin/CHO-K1 cells with median fluorescence intensity (MFI) of 3.23E5; the red curve corresponds to parental CHO-K1 control cells with median fluorescence intensity of 2457. The obvious separation of two peaks demonstrates high and specific expression of CCR8 receptor on the constructed stable cell line.

Figure 2. Dose response of Human Recombinant CCL1 in CCR8 β-Arrestin

Recombinant human CCL1 was applied as agonist to trigger receptor-mediated β-arrestin recruitment signal. The dose-response curve was plotted with RLU value against log concentration of hCCL1. The EC50 value of hCCL1 on this cell line was determined as 6.29 ng/ml, verifying that the expressed CCR8 receptor can effectively activate β-arrestin signaling pathway with good biological activity.

Figure 3. Inhibition of  rhCCL1-induced β-Arrestin Recruitment by CCR8 Blocking Ab in CCR8 β-Arrestin CHO(C6C6)

The experiment detects the inhibitory effect of BMS-986340 on rhCCL1-triggered β-arrestin recruitment. The inhibition dose-response curve was plotted with percentage inhibition against log concentration of BMS-986340. The IC50 value was determined as 102.4 ng/ml. The result demonstrates that this cell line is suitable for antagonist and blocking antibody functional screening targeting human CCR8.

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 CCR8 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.