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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, which constitute bone tissue. Membrane-bound RANKL on osteoblasts binds to RANK on the surface of osteoclast precursors to stimulate osteoclastogenesis, bone remodeling, and calcium homeostasis.

The cytoplasmic region of RANK lacks intrinsic kinase activity and requires adaptor molecules for downstream signal transduction. Upon activation by RANKL, the RANK receptor trimerizes and recruits TNF 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 or excessive bone density. RANKL/RANK binding activates downstream intracellular signaling pathways—including MAPK, NF-κB, and PI3K—via TRAF6; this leads to increased expression of NFATc1, thereby promoting osteoclastogenesis and bone resorption.

Furthermore, RANKL plays a role in specific types of cancer; in osteosarcoma, in addition to mediating cancerous bone destruction, RANKL is implicated in tumor initiation and metastasis. Studies using mouse models of breast cancer have also demonstrated that the inhibition of RANKL can significantly delay the formation of carcinogen- and hormone-induced breast tumors.

Figure 1.  Recombinant Membrane Anchored hRANKL Cell stably expressing RANKL.

Figure 2. Dose response of Anti-hRANKL-hIgG1in ADCC Bioassay Effector Cell V variant (High Affinity)-Fcγ-NFAT Jurkat with Membrane Anchored RANKL Cell (C12).

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  CHO-K1 Human Membrane Anchored hRANKL 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.