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Figure 1. HTRF cAMP assay with ADORA2A Gα15 CHO-K1 (C1)

The ADORA2A Gα15 CHO-K1 (C1) cell line was functionally validated by HTRF cAMP assay. NECA and adenosine induced dose-dependent cAMP responses through ADORA2A-Gα15 coupling, with EC50 values of 11 nM and 250 nM respectively, confirming potent, functional receptor coupling suitable for high-throughput screening and QC lot release.

Figure 2. HTRF cAMP assay in ADORA2A Gα15 CHO-K1 (C1) stimulated with NECA

The ADORA2A Gα15 CHO-K1 (C1) cell line was functionally validated by HTRF cAMP antagonist assay. Imaradenant and Ciforadenant produced dose-dependent inhibitory responses against NECA-stimulated receptor activation, with IC50 values of 37.4 nM and 23.9 nM respectively, confirming potent receptor antagonism suitable for high-throughput screening and QC lot release.

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 ADORA2A Gα15 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.