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The IL-1 family comprises 11 cytokines, consisting of 7 ligands with agonist activity (IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β, and IL-36γ) and 4 members with antagonist activity [IL-1 Receptor Antagonist (IL-1Ra), IL-36Ra, IL-37, and IL-38]. IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β, and IL-36γ trigger the activation of MAP kinases and NF-κB, thereby inducing an inflammatory response.

Similar to other cytokine members of the IL-1 family, IL-36 agonists (IL-36α, β, and γ) bind to a heterodimeric receptor complex composed of the IL-36 receptor (IL-36R) and the IL-1 receptor accessory protein (IL-1RAcP). IL-36R is expressed by numerous cell types, including keratinocytes, pulmonary fibroblasts and epithelial cells, endothelial cells, and various immune cells. IL-36R is also highly expressed in human M0 and M2 macrophages, though it is not highly expressed in M1 macrophages. IL-36Ra and IL-38 exert negative regulatory effects on the IL-36 signaling pathway. IL-36Ra functions as an antagonist by competitively binding to IL-36R, thereby inhibiting the recognition of IL-36 agonists and the recruitment of IL-1RAcP; this action effectively blocks the receptor activation induced by the agonist members of this family. In addition to blocking the IL-36R-mediated activation of the MAP kinase and NF-κB pathways, IL-36Ra can also suppress the expression of Th17 cytokines.

The IL-36 Effector Reporter Cell (Adherent) model—a reporter gene-based drug target model—accurately simulates the in vivo signal transduction processes of IL-36; the underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the IL36 Effector Reporter Cell(Adherent) Cell Model

Figure 2. Recombinant IL36 Effector Reporter Cell(Adherent) stably expressing IL36R.

Figure 3. Dose Response of Recombinant Human IL36 alpha Protein in IL36 Effector Reporter Cell(Adherent, C12).

Figure 4. Inhibition of hIL36α Induced Reporter Activity By Blocking Abs in IL36 Effector Reporter Cell(Adherent, C12).

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  IL36 Effector Reporter Cell(Adherent) 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.