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IL-31 belongs to the IL-6-derived cytokine family. Cytokines of the IL-6 family are typically classified based on their pro-inflammatory properties and their involvement in common signaling pathways that activate the GP130 receptor. The IL-6 cytokine family is often referred to as the GP130/IL-6 cytokine family. Members of this family include IL-6, IL-11, IL-21, IL-27, Neuropoietin (NPN), Ciliary Neurotrophic Factor (CNTF), Cardiotrophin-1 (CT-1), Leukemia Inhibitory Factor (LIF), Oncostatin M (OSM), and IL-31. Members of this family share the GP130 subunit within their multi-subunit receptors and are involved in neuronal growth, bone metabolism, cardiac development, and immune regulation—such as T-cell differentiation. IL-31 is unique within this cytokine family because its receptor complex does not contain GP130.

IL-31 signals through a heterodimeric receptor complex composed of the IL-31 receptor α (IL-31RA) and Oncostatin M receptor β (OSMRβ) subunits. Within the IL-31R complex, IL-31 primarily binds to IL-31RA, while it does not bind to OSMRβ. However, upon association, OSMRβ converts IL-31RA into a high-affinity receptor and enhances the binding of IL-31. Binding to the receptor complex leads to the activation of Janus kinase (JAK) tyrosine kinases, which subsequently triggers the activation of various signaling molecules—including different Signal Transducers and Activators of Transcription (STAT) factors—as well as the MAPK and PI3K signaling pathways.

The HEK293 Canine IL31 Effector Reporter Cell Model—effectively simulates the signal transduction process of IL31 *in vivo*. The underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of theHEK293 Canine IL31 Effector Reporter Cell  Model

Figure 2. Dose Response of Recombinant Canine IL31 in Canine IL31 Effector Reporter Cell (C10).

Figure 3. Inhibition of Canine IL31-induced Reporter Activity by Canine IL31 Neutralization Ab in Canine IL31 Effector Reporter Cell(C10).

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 Canine IL31 Effector Reporter Cell 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.