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Glucagon is a pancreatic peptide hormone that acts as a counterregulatory hormone to insulin, stimulating the release of glucose through the liver to maintain glucose homeostasis. GCGR is a class B GPCR, initially described as a glucagon-binding enzyme linked to adenylate cyclase function. It plays a crucial role in maintaining glucose homeostasis and is therefore considered a valuable target for diabetes treatment.

GCGR's natural ligand is glucagon. Glucagon binds to its receptor and activates the Gs protein, thereby activating adenylate cyclase. The binding of the GCGR receptor to its natural ligand, glucagon, regulates glucose homeostasis and is an important drug target for type 2 diabetes.

The HEK293 Human GCGR CRE-Luc Cell Line model serves as an excellent mimic of the in vivo GCGR signal transduction process; the underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the HEK293 Human GCGR CRE-Luc Cell Line Model

Figure 2. Recombinant GCGR/CRE-Luc/HEK293 stably expressing GCGR.

Figure 3. HTRF cAMP Assay with GCGR CRE-Luc HEK293( C21).

Figure 4. Dose Response of Ligands in GCGR CRE-Luc HEK293(C21).

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 HEK293 Human GCGR CRE-Luc 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.