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Figure 1. Recombinant CRTH2(DP2)/CHO-K1 constitutively expressing CRTH2(DP2)

The surface expression of CRTH2 was detected by anti-CRTH2 antibody. The mean fluorescence intensity (MFI) of CRTH2(DP2)/CHO group reached 3.60E6, while the parental CHO cell control group was 1098. The obvious peak shift demonstrates high and stable expression of CRTH2 receptor on cell membrane.

Figure 2. HTRF CAMP Assay with CRTH2(DP2) CHO-K1 Cell (C1)

Intracellular cAMP level was detected by HTRF assay, with EC50 = 2.5 nM. The curve demonstrates that agonist stimulation activates CRTH2 receptor and modulates intracellular cAMP concentration, verifying intact G-protein signaling function of this cell line for GPCR compound screening.

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) 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.