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 The IL-10 (interleukin-10) family of cytokines includes several members, such as IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-28A/B, and IL-29, all of which belong to the class 2α helical cytokines. IL-19, IL-20, IL-22, IL-24, and IL-26 also belong to the IL-20 subfamily. The IL-20 subfamily is a key driver of skin inflammation (particularly in psoriasis) and is also involved in Th2 diseases (asthma, atopic dermatitis) and the regulation of the tumor microenvironment.

      There are four heterodimeric receptors that serve as functional receptors for cytokines in the IL-20 family, and IL-24 signaling is transduced through two distinct heterodimeric receptors. One IL-24 receptor is the type I IL-20 receptor (IL-20R), composed of IL-20RA and IL-20RB; this receptor shares IL-19 and IL-20 as ligands. IL-24 and IL-20 can also transmit additional signals through the type II IL-20R, which consists of IL-20RB and the IL-22 receptor subunit (IL-22RA1). The receptors for IL-22 and IL-26 consist of IL-22RA1 and IL-20RA, respectively, and both receptors share IL-10RB. Both type I and type II IL-20 receptors are expressed on non-immune cells, such as keratinocytes and bronchial epithelial cells, but not on immune cells. Therefore, it is believed that IL-24 cannot activate immune cells.

  The hIL20/IL22/IL24 Triple Effector Reporter Cell model effectively mimics the in vivo signaling pathways of IL20, IL22, and IL24; the underlying mechanism is illustrated in the figure below.

Figure 1. Schematic diagram of the  hIL20/IL22/IL24 Triple Effector Reporter Cell model

Figure 2. Recombinant hIL20/IL22/IL24 Triple Effector Reporter Cell stably expressing IL22R1.

Figure 3. Dose Response of Ligands in hIL20/IL22/IL24 Triple Effector Reporter Cell(C30).
 

Figure 4. Inhibition of Human IL22-induced Reporter Activity by IL22Ra Blocking Ab or IL22 Neutralization Ab in hIL20/IL22/IL24 Triple Effector Reporter Cell(C30).
 

Figure 5. Inhibition of Human IL20-induced Reporter Activity by IL20 Neutralization Ab in hIL20/IL22/IL24 Triple Effector Reporter Cell(C30).

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 IL20 IL22 IL24 Triple 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.