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Hypocretin—also known as orexin—refers to two neuropeptides synthesized and secreted by specific neurons in the lateral hypothalamus: Hypocretin-1 (Orexin A) and Hypocretin-2 (Orexin B). These neuropeptides project extensively throughout the central nervous system, regulating the sleep-wake cycle, feeding behavior, mood, energy metabolism, and autonomic functions by activating their corresponding G protein-coupled receptors (OX1R and OX2R).

OX2R (Orexin Receptor Type 2, also known as Hypocretin Receptor Type 2) is a high-affinity receptor for hypocretins; it is encoded by the *HCRTR2* gene and belongs to the family of G protein-coupled receptors. Unlike OX1R, OX2R binds indiscriminately to both Hypocretin-1 and Hypocretin-2. It is widely distributed across various brain regions—including the hypothalamus, cortex, thalamus, and nucleus accumbens—while its expression in peripheral tissues is extremely low. Upon the binding of hypocretin to the extracellular domain of OX2R, the receptor undergoes a conformational change, thereby transducing extracellular chemical signals into intracellular signaling cascades. Given the dominant role of OX2R in maintaining wakefulness, functional deficits in this receptor or the loss of hypocretin-producing neurons can lead to narcolepsy (Type 1); conversely, excessive activation may disrupt sleep. Consequently, activating OX2R via agonists offers a therapeutic strategy for hypersomnia-related disorders, whereas blocking OX2R with antagonists can induce sleep and treat insomnia.

The CHO-K1 Human OX2R Cell Line model effectively simulates the in vivo OX2R signal transduction process, the principle is illustrated in the figure below.

Figure 1. Schematic diagram of the CHO-K1 Human OX2R Cell Line model

Figure 2. HTRF IP-One Assay with OX2R CHO(C20).

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 OX2R 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.