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Glucagon is a pancreatic peptide hormone that acts as a counter-regulatory hormone to insulin; it stimulates hepatic glucose release to maintain glucose homeostasis. GCGR is a Class B GPCR, originally characterized as a glucagon-binding protein functionally linked to adenylate cyclase. It plays a pivotal role in maintaining glucose homeostasis and is therefore considered a valuable therapeutic target for diabetes.

Glucagon is the endogenous ligand for GCGR. Binding of glucagon to the receptor activates Gs proteins, which in turn activate adenylate cyclase. The interaction between GCGR and its endogenous ligand, glucagon, regulates glucose homeostasis in the body, making it a significant drug target for type 2 diabetes.

cAMP serves as a key second messenger in G protein-coupled receptor (GPCR) signaling. Ligand binding induces a conformational change in the GPCR, activating the receptor and subsequently the G protein; activation of Gs leads to adenylate cyclase-mediated elevation of cAMP levels. The mouse GCGR CHO drug target model effectively recapitulates the in vivo GCGR signaling process; the underlying mechanism is illustrated in the figure below.

Figure 1. Schematic diagram of the mouse GCGR CHO cell model.

Figure 1. HIRF cAMP Assay with Mouse GCGR CHO-K1(C10).

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 Mouse GCGR 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.