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Glucagon is a pancreatic peptide hormone that acts as a counter-regulatory hormone to insulin; it stimulates the release of glucose from the liver, thereby maintaining glucose homeostasis. The GCGR is a Class B GPCR, originally characterized as a glucagon-binding entity functionally coupled to adenylyl cyclase. It plays a pivotal role in maintaining glucose homeostasis and is, therefore, considered a valuable therapeutic target for the treatment of diabetes.

The endogenous ligand for the GCGR is glucagon. Upon binding to the receptor, glucagon activates the Gs protein, which in turn activates adenylyl cyclase. The binding of the GCGR to its endogenous ligand, glucagon, regulates glucose homeostasis *in vivo*, making it a critical drug target for Type 2 diabetes.

The GCGR/β-Arrestin 1 CHO reporter gene assay model accurately mimics the *in vivo* signal transduction process of the GCGR/β-Arrestin 1 pathway; the underlying mechanism is illustrated in the figure below. In the absence of ligand stimulation, β-Arrestin 1 does not bind to the GCGR, and the luciferase fused to β-Arrestin 1 remains in an inactive conformation. However, upon GCGR stimulation by a ligand, the β-Arrestin 1—carrying the fused luciferase reporter gene—is recruited to the receptor; this shifts the luciferase reporter gene into an active state, resulting in an enhanced luminescent signal following the addition of its substrate.

Figure 1. Schematic Diagram of the GCGR β-Arrestin 1 CHO Cell Model

Figure 1.  Recombinant GCGR β-Arrestin 1 CHO-K1 constitutively expressing GCGR.

Figure 2. Dose Response of Glucagon(1-29) in GCGR β-Arrestin 1 CHO-K1 (C12).

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 GCGR β-Arrestin 1 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.