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The Wnt signaling pathway is an evolutionarily conserved pathway that governs multiple aspects of development, including cell proliferation, apoptosis, cell migration, cell polarity, and stem cell maintenance during adult development.  

Low-density lipoprotein (LDL) receptor-related proteins 5 and 6 (LRP5/6) are single-pass transmembrane glycoproteins that serve as essential co-receptors for the canonical Wnt signaling pathway. LRP5/6 possesses an extracellular domain (ECD) composed of four repeating units (E1, E2, E3, and E4); this ECD acts as a critical regulatory site for Wnt signaling, providing at least two independent binding sites for Wnt ligands. Activation of the canonical Wnt signaling pathway is initiated when a Wnt ligand binds to its co-receptor LRP5/6, subsequently forming a complex with a Frizzled (FZD) receptor. Upon the formation of the Wnt-FZD-LRP5/6 complex, the Axin-mediated destruction complex is recruited to the plasma membrane—facilitated by phosphorylation events involving other proteins within the destruction complex—thereby inhibiting the phosphorylation of β-catenin. This destruction complex consists of Axin, APC, CK1, and GSK3.  

The Wnt Effector Reporter Cell model serves as a robust drug-target platform that accurately mimics the in vivo Wnt signal transduction process; the underlying principle is illustrated in the figure below.

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

Figure 2. Dose Response of Recombinant Human Wnt-3a in Wnt Effector Reporter Cell (C2).

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 Wnt 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.