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Tumor Necrosis Factor (TNF) Receptor Superfamily Member 11a (TNFRSF11a, also known as RANK) is a type I transmembrane protein found on osteoclast precursors, mature osteoclasts, and immune cells. RANKL is a type II transmembrane protein expressed by osteoblasts, osteocytes, and immune cells—the cellular components that constitute bone tissue. Membrane-bound RANKL on osteoblasts binds to RANK on the surface of osteoclast precursors, thereby stimulating osteoclastogenesis, bone remodeling, and calcium homeostasis.

The cytoplasmic region of RANK lacks intrinsic kinase activity and requires adaptor molecules to mediate downstream signal transduction. Upon activation by RANKL, the RANK receptor trimerizes and recruits Tumor Necrosis Factor Receptor-Associated Factors (TRAFs). Although TRAF2, TRAF5, and TRAF6 are all capable of binding to the cytoplasmic domain of RANK, only mutations in TRAF6 result in osteopetrosis—a condition characterized by excessive bone density. RANKL/RANK binding activates downstream intracellular signaling pathways—including MAPK, NF-κB, and PI3K—via TRAF6; this activation leads to increased expression of NFATc1, thereby promoting osteoclastogenesis and bone resorption.

Furthermore, RANKL plays a role in specific types of cancer. In osteosarcoma, for instance, RANKL is implicated in both tumor initiation and metastasis, in addition to its involvement in cancer-associated bone destruction. Studies utilizing mouse models of breast cancer have also demonstrated that the inhibition of RANKL can significantly delay the formation of carcinogen- and hormone-induced mammary tumors.

The RANK Effector Reporter Cell model serves as an excellent *in vitro* system for simulating the signal transduction processes of RANK as they occur *in vivo*; the underlying principle is illustrated in the figure below.

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

Figure 2. Dose response of  Recombinant Human RANKL Protein in RANK Effector Reporter Cell (C31).

Figure 3. lnhibition of rhRANKL-induced Reporter Activity by RANKL Neutralizing Antibody in RANK Effector Reporter Cell(C31).

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 RANK 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.