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Figure 1.Recombinant CCR5/CHO-K1 stably expressing CCR5

Flow cytometry analysis of recombinant CCR5/CHO-K1 stable cell line for CCR5 receptor expression. Blue curve: CCR5/CHO-K1 cells stained with anti-CCR5 antibody, Mean FL4-A = 2.66E6. Red curve: Parental CHO-K1 negative control cells stained with anti-CCR5 antibody, Mean FL4-A = 1892. Significant fluorescence shift confirms high membrane expression of human CCR5 receptor

Figure 2. HTRF cAMP Assay with CCR5 CHO(C4)

The HTRF ratio was measured to characterize rhCCL4 induced cAMP response, demonstrating rhCCL4 activates CCR5 in a dose-dependent manner

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 CCR5 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.