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Figure 1. Recombinant CCR2/β-Arrestin/CHO-K1 constitutively expressing CCR2

Flow cytometry analysis of human CCR2 expression on CCR2/β-Arrestin CHO-K1 stable cell line. The CCR2/β-Arrestin/CHO-K1 cells (blue curve) exhibited high fluorescence intensity with Mean FL4-A = 4.09E6, while the β-Arrestin/CHO-K1 control cells (red curve) showed minimal signal (Mean FL4-A = 1610). The result verifies robust and stable membrane expression of human CCR2 receptor.

Figure 2. Dose response of Human CCL2 in CCR2 β-Arrestin CHO-K1(C10)

The CCR2 β-Arrestin/CHO-K1 (C10) stable cell line was functionally characterized by β-Arrestin recruitment luminescence assay. Human CCL2 induced a robust dose-dependent response with EC50 = 6 ng/ml and signal-to-background (S/B) ratio of 5.6, verifying specific β-Arrestin recruitment activity of human CCR2 receptor, suitable for high-throughput screening and pharmacological evaluation.

Figure 3. Dual inhibition curves of hCCL2-induced β-Arrestin recruitment in CCR2 β-Arrestin CHO-K1 (C10) cell line

Left: CCL2 neutralizing antibody (Carlumab) exhibited inhibitory activity with IC50 = 0.43 μg/ml. Right: Small molecule antagonist PF-4136309 showed potent inhibition with IC50 = 17.8 nM. This assay system is suitable for screening and potency evaluation of CCR2 antagonists and neutralizing antibodies.

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 CCR2 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.