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Figure 1.Recombinant CRTH2(DP2) β-Arrestin CHO-K1 stably expressing CRTH2(DP2)

CRTH2 expression was detected using anti-CRTH2 antibody. The median fluorescence intensity (MFI) of CRTH2(DP2) β-Arrestin CHO(C15) group reached 3.11E5, while the parental cell control group was 517, which demonstrates high and stable membrane expression of CRTH2.

Figure 2. Dose Response of Prostaglandin D2 in CRTH2(DP2) β-Arrestin CHO-K1(C15)

The β-arrestin recruitment signal was measured, with EC50 = 48.51 nM. The result confirms that CRTH2 receptor can effectively recruit β-arrestin upon agonist stimulation, verifying the biological function of this cell line.

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 CRTH2(DP2) 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.