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The RXFP (Relaxin Family Peptide Receptor) family is a class of G protein-coupled receptors comprising four members: RXFP1, RXFP2, RXFP3, and RXFP4. RXFP2—also known as LGR8 or GREAT—features a protein structure characterized by seven transmembrane domains, an extracellular leucine-rich repeat region, and a Class A low-density lipoprotein (LDL) module; its primary physiological ligand is Insulin-like Peptide 3 (INSL3).

During the fetal period, the binding of INSL3 to RXFP2 activates downstream signaling pathways (such as increasing intracellular cAMP levels), thereby driving the process of transabdominal testicular descent; functional impairment of this mechanism results in bilateral cryptorchidism. Upon the onset of puberty, RXFP2 expression levels rise in tandem with increasing testosterone levels, and the receptor participates directly in spermatogenesis and sperm maturation—playing an indispensable role specifically during the differentiation of round spermatids into mature spermatozoa. Impaired function of this receptor can lead to spermatogenic arrest and non-obstructive azoospermia. Furthermore, RXFP2 may also be involved in other physiological processes, such as immune modulation and bone metabolism.

The CHO-K1 Human RXFP2 [K658E] Cell Line model effectively simulates the in vivo RXFP2 [K658E] signal transduction process, the principle is illustrated in the figure below.

Figure 1. Schematic diagram of the CHO-K1 Human RXFP2 [K658E] Cell Line model

Figure 2. HTRF cAMP Assay with RXFP2 CHO(C57) in 500 μM IBMX.

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 RXFP2 [K658E] 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.