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Growth Differentiation Factor 15 (GDF15), also known as Macrophage Inhibitory Cytokine 1 (MIC-1), belongs to the TGF-β superfamily of proteins. GDF15 is an endocrine hormone primarily involved in cell growth, differentiation, and tissue repair. As an inflammatory marker, GDF15 plays a role in the pathogenesis of tumors, ischemic diseases, metabolic disorders, and neurodegenerative processes.

      GFRAL (GDNF-family receptor α-like), also known as GRAL, belongs to the glial cell-derived neurotrophic factor (GDNF) family of α-like receptors. GFRAL is a transmembrane protein expressed exclusively in the hindbrain and requires the co-receptor RET for signal transduction. When GDF15 binds to GFRAL, RET undergoes autophosphorylation, leading to the activation of downstream signaling pathways (such as PI3K-AKT and PLC-PKC), thereby regulating numerous physiological processes, including influencing food intake and causing weight loss. Inhibiting the GDF15-GFRAL signaling pathway enhances the immune system’s ability to kill solid tumors and can treat or prevent cancer cachexia.

The GDF/GFRAL&RET Effector Reporter Cell model effectively mimics the in vivo GDF signaling pathway; the mechanism is illustrated in the figure below.

Figure 1. Schematic diagram of the GDF/GFRAL&RET Effector Reporter cell model

Figure 2. Recombinant GDF/GFRAL&RET Effector Reporter Cell constitutively expressing Ret.

Figure 3. Recombinant GDF/GFRAL&RET Effector Reporter Cell constitutively expressing GFRAL.

Figure 4.Dose Response of Recombinant Human GDF-15 in GDF/GFRAL&RET Effector Reporter Cells (C1).

Figure 5.Inhibition of GDF-15-induced Reporter Activity by GDF-15 Neutralization Ab in GDF/GFRAL&Ret Effector Reporter Cell (C1).

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 GDF/GFRAL&RET 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.