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NPFFR2 (Neuropeptide FF Receptor 2) is a seven-transmembrane protein belonging to the GPCR family. It is widely distributed throughout the central and peripheral nervous systems, with particularly high expression levels observed in brain regions closely associated with pain perception, stress response, and the regulation of energy homeostasis—such as the spinal cord, hypothalamus, and amygdala. In addition to its natural ligand, Neuropeptide FF, NPFFR2 can also be activated by various endogenous members of the RF-amide peptide family, thereby establishing a complex signaling network. Given its dual role in pain modulation and the regulation of opioid function, NPFFR2 has remained a pivotal research target in the fields of analgesia and addiction since its discovery. In recent years, associations between genetic variants of this receptor and susceptibility to conditions such as preeclampsia, obesity, diabetes, and autism spectrum disorders have gradually come to light, establishing it as a focal point of research in the fields of neurometabolism and psychiatric disorders.

The HEK293 Human NPFFR2 NFAT-Luc Cell Line model effectively simulates the in vivo NPFFR2 NFAT-Luc signal transduction process, the principle is illustrated in the figure below.

Figure 1. Schematic diagram of the HEK293 Human NPFFR2 NFAT-Luc Cell Line model

Figure 2. Detect Luciferase assay by Ultra Luciferase Detection Kit RQPH0001. Dose Response of Agonists in NPFFR2 NFAT-Luc HEK293(C38).

Figure 3. HTRF cAMP Assay with NPFFR2 NFAT-Luc HEK293 (C38).

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 NPFFR2 NFAT-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.