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MC2R (melanocortin-2 receptor) is a member of the G protein-coupled receptor family that is specifically expressed in the adrenal cortex and serves as the sole functional receptor for ACTH (adrenocorticotropic hormone). Upon binding to MC2R, ACTH activates the cAMP signaling pathway, stimulating the synthesis and release of glucocorticoids (such as cortisol), which are critical for stress responses and metabolic regulation. Mutations in the MC2R gene can lead to Familial Glucocorticoid Deficiency (FGD).

The CHO-K1 Human MC2R CRE-Luc Cell Line Model—effectively simulates the signal transduction process of MC2R *in vivo*.  

Figure 1. Detect Luciferase assay by Ultra Luciferase Detection Kit RQPH0001（we strongly suggest to purchase from Cobioer). Dose Response of Human ACTH(1-39) in MC2R CRE-Luc CHO(C16).

Figure 2. Dose Response of ACTH in MC2R CRE-Lue CHO(C16).

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 MC2R 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.