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Interleukin-5 (IL-5) is a member of the Th2 cytokine family (Type 2 inflammatory factors); it is primarily secreted by activated Th2 cells, mast cells, and eosinophils, and plays a pivotal role in regulating the generation, activation, and survival of eosinophils. IL-5 is currently recognized as one of the key drivers of the Th2 pathway and plays a significant role in autoimmune diseases.

The IL-5 receptor consists of a specific α-chain (IL-5Rα) and a non-specific βc-chain (which is shared with the IL-3 and GM-CSF receptors). Upon binding to IL-5Rα, IL-5 associates with the βc-chain to form an IL-5/IL-5Rα/βc complex; this activates intracellular JAK kinases, which in turn activate the signal transducers and activators of transcription (STAT) 1, 3, and 5, thereby inducing the transcription of numerous genes involved in the proliferation and activation of eosinophils. JAK2—along with JAK1, which is associated with the βc-chain—can simultaneously activate the Raf-1 and NF-κB signaling pathways, thereby influencing the apoptotic processes of eosinophils. The signaling pathways activated by IL-5 also include the PI3K and MAPK pathways, which mediate the recruitment, maturation, differentiation, and proliferation of eosinophils.

The HEK293 Human IL5 Effector Reporter Cell Model—effectively simulates the signal transduction process of IL5 *in vivo*. The underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the HEK293 Human IL5 Effector Reporter Cell Model

Figure 2. Recombinant hIL5 Effector Reporter Cell constitutively expressing IL5Rα.

Figure 3. Dose Response of Recombinant Human IL5 in IL5 Effector Reporter Cell (C9).

Figure 4. Inhibition of hIL5-induced Reporter Activity by Abs in IL5 Effector Reporter Cell (C9).

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 IL5 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.