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Tachykinins are a class of neuropeptides distributed throughout the mammalian central and peripheral nervous systems. The tachykinins Substance P (SP), Neurokinin A (NKA), and Neurokinin B (NKB) share a conserved C-terminal motif, which is critical for the activation of tachykinin receptors (NK1R, NK2R, and NK3R). NKA preferentially activates the Gα-coupled NK2R, while SP and NKB preferentially bind to NK1R and NK3R, respectively. All three tachykinins are capable of acting as full agonists across all tachykinin receptor subtypes.  

The NK1R (Neurokinin 1 Receptor)—also known as TACR1—is a core member of the tachykinin receptor family, with Substance P serving as its endogenous ligand. As a pleiotropic GPCR target, NK1R plays a pivotal role in pain transmission, inflammatory responses, and mood regulation; consequently, it represents a key therapeutic target for antiemetic, antidepressant, and analgesic medications.  

The HEK293 Human NK1R CRE-Luc Cell Line Model—effectively simulates the signal transduction process of NK1R *in vivo*. The underlying principle is illustrated in the figure below.  

Figure 1. Schematic Diagram of the HEK293 Human NK1R CRE-Luc Cell Line Model

Figure 2.Dose Response of Agonists in NK1R CRE-Luc HEK293(C1).

Figure 3. HTRF cAMP Assay with NK1R CRE-Luc HEK293(C1) in 500 μM IBMX.

Figure 4. HTRF IP-One Assay with NK1R CRE-Luc HEK293(C1).

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 NK1R 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.