Rankl

Manufactured by R&D Systems

Sourced in United States, United Kingdom, China, Japan, Germany, Israel

RANKL is a recombinant protein produced in mammalian cells. It is a member of the tumor necrosis factor (TNF) ligand family and plays a key role in the regulation of bone remodeling and immune function.

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Lab products found in correlation

M csf, by R&D Systems (225 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (84 mentions) α mem, by Thermo Fisher Scientific (44 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (37 mentions) Trap staining kit, by Merck Group (24 mentions) Leukocyte acid phosphatase kit, by Merck Group (23 mentions) Penicillin, by Thermo Fisher Scientific (23 mentions) M csf, by Thermo Fisher Scientific (19 mentions) Streptomycin, by Thermo Fisher Scientific (19 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (15 mentions) Recombinant mouse m csf, by R&D Systems (12 mentions) Macrophage colony stimulating factor m csf, by R&D Systems (11 mentions) Recombinant m csf, by R&D Systems (11 mentions) β actin, by Cell Signaling Technology (11 mentions) α mem, by R&D Systems (10 mentions) Dimethyl sulfoxide (dmso), by Merck Group (10 mentions) Tnf α, by R&D Systems (9 mentions) P erk, by Cell Signaling Technology (9 mentions) Dexamethasone (dex), by Merck Group (9 mentions) Ascorbic acid, by Merck Group (9 mentions)

491 protocols using rankl

Osteoclast Differentiation from RAW264.7 and BMMs

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RAW264.7 cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured in alpha-minimum essential medium (α-MEM; Gibco, New York, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) at 37 °C under an atmosphere with 5% CO₂. Mouse bone marrow cells were isolated from the long bones of 6- to 8- week-old C57BL/6 mice (Animal Center of Sun Yat-sen University) by flushing the bone marrow cavity with α-MEM. Then, the cells were cultured in a-MEM containing 10% FBS overnight to separate the suspended cells. The suspended cells were then collected and cultured in α-MEM containing 10% FBS with 30 ng/mL M-CSF (Sino Biological, Beijing, China) for 3 days to obtain BMMs, which were used as osteoclast precursor cells.
For osteoclast differentiation, RAW264.7 cells were stimulated with 50 ng/mL RANKL (R&D Systems, Minneapolis, MN, USA) for 5 days, while BMMs were treated with 30 ng/mL M-CSF and 50 ng/mL RANKL for 5 days. The culture medium was changed every two days. Cells treated without RANKL served as a negative control. Mature osteoclasts were detected by TRAP staining.

Li D., Cai L., Meng R., Feng Z, & Xu Q. (2020). METTL3 Modulates Osteoclast Differentiation and Function by Controlling RNA Stability and Nuclear Export. International Journal of Molecular Sciences, 21(5), 1660.

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Osteoclast Differentiation from Bone Marrow Macrophages

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Bone marrow macrophages (BMMs) were plated at a density of 1 × 10 4 cell/well with macrophage colony-stimulating factor (M-CSF, 30 ng/ml) and commercial RANKL (R&D Systems, MN, USA) or the purified RANKL in our laboratory. Cells were incubated for 4 days at 37°C, 5% CO 2 . After 4 days, cells were fixed with 3.7% formalin for 5 min, permeabilized with 0.1% Triton X-100 for 10 min, and stained with TRAP solution (Sigma-Aldrich, MO, USA) for 10 min. To check the biological activity of rRANKL, multinucleated osteoclasts were observed under a microscope.

Park S.J., Lee S.H., Kim K.J., Kim S.G., Kim H., Choe H., Lee S.Y., Yun J.M., Cho J.Y., Chun J., Choi K.S, & Son Y.J. (2015). Soluble expression and purification of receptor activator of nuclear factor-kappa B ligand using Escherichia coli. Journal of microbiology and biotechnology, 25(2).

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HDAC7 and MITF Regulation of Osteoclastogenesis

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Bone marrow macrophages were isolated as described above. Prior to stimulation with RANKL, the cells were incubated with 100 MOI of adenovirus (EGFP control, CRE recombinase, HDAC7 wild type or HDAC7^D692A/694A) for 3 hours at 37°C in the presence of 1% CMG 14–12 culture supernatant. After 3 hours, the media containing adenovirus was removed and cells were fed with 1% CMG 14–12 culture supernatant and RANKL (30 ng/mL, R&D Systems). After five days, RNA was extracted for use in qRT-PCR, protein was extracted for western blotting, or cells were stained for TRAP.
Lentiviral vectors encoding shRNAs against Mitf (Open Biosystems) or a control shRNA were used to produce replication defective lentivirus according to the manufacturer’s protocols. Viral stocks were titrated by infection in HeLa cells. Bone marrow macrophages were isolated as described above. Forty-eight hours after M-CSF stimulation, lentiviruses were added and incubated for 18 hours at 37°C in the presence of 1% CMG 14–12 culture supernatant. The lentivirus-containing supernant was then removed and cells were fed with 1% CMG 14–12 culture supernatant and RANKL (30 ng/mL, R&D Systems). After five days, RNA was extracted for use in qRT-PCR or cells were stained for TRAP.

Stemig M., Astelford K., Emery A., Cho J.J., Allen B., Huang T.H., Gopalakrishnan R., Mansky K.C, & Jensen E.D. (2015). Deletion of Histone Deacetylase 7 in Osteoclasts Decreases Bone Mass in Mice by Interactions with MITF. PLoS ONE, 10(4), e0123843.

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Osteoclast Differentiation in RAW 264.7 Cells

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Murine monocyte/macrophage RAW 264.7 cells (Sumitomo Dainippon Pharma, Osaka, Japan) were grown in minimal essential medium alpha (Nacalai Tesque) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Biowest, Nuaille, France), 100 U/mL penicillin, and 100 μg/mL streptomycin (Wako Pure Chemical Industries, Osaka, Japan) at 37°C in a humidified atmosphere of 5% CO₂ in air. For differentiation of osteoclasts, RAW 264.7 cells (5,000 cells per well in a 96-well plate) were cultured in the presence of 10 ng/mL receptor activator of nuclear factor kappa B ligand (RANKL; R&D Systems) for 2 days, and then the cells were stimulated with 10 ng/mL RANKL and 10 ng/mL recombinant human TNF-α (R&D Systems) in the presence or absence of the drugs for 3 days.

Seki I., Takai-Imamura M., Kohara-Tanaka T., Shirae S., Sasano M., Matsuno H, & Aono H. (2019). Concomitant Treatment with Etanercept and Tacrolimus Synergistically Attenuates Arthritis Progression via Inhibition of Matrix Metalloproteinase-3 Production and Osteoclastogenesis in Human TNF-α Transgenic Mice. Mediators of Inflammation, 2019, 4176974.

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Osteoclast Differentiation from Bone Marrow Macrophages

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To induce osteoclast differentiation, 1 × 10⁴ BMMs were inoculated into each well of 96 well plate. Add 30 ng/mL M-CSF and 50 ng/mL RANKL (R&D Systems) as osteoclast culture medium, and set it as RANKL group.
After 5 days of culture, the osteoclasts were stained with TRAP according to the instructions of TRAP Kit (Sigma-Aldrich, USA). The images of TRAP-positive cells with nuclei ≧ 3 were obtained using a light microscope (Zeiss, Germany) and the area of TRAP-positive osteoclasts were counted in six randomly chosen fields of view.

Cao Z., Xue Y, & Wang J. (2023). Screening diagnostic markers of osteoporosis based on ferroptosis of osteoblast and osteoclast. Aging (Albany NY), 15(18), 9391-9407.

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Calcium Imaging of Bone Cells

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A total of 1 × 10⁴ BMMs or human PBMCs were seeded on 35-mm confocal dishes and cultured with 30 ng/mL M-CSF (R&D Systems, Minneapolis, MN, USA) in the presence of 50 ng/mL RANKL (R&D Systems, Minneapolis, MN, USA) or without RANKL as controls for 72 h. BMSCs (2 × 10⁴) were cultured on confocal dishes with osteogenic media for 7 days. The assay buffer consisted of Hank’s buffered salt solution (Gibco, Gaithersburg, MD, USA, without CaCl₂), 2% FCS, and 1 mM probenecid solution (Invitrogen, Carlsbad, CA, USA) (pH 7.4). The cells were then incubated in the presence of 5 µM Fluo-4 AM (Invitrogen, Carlsbad, CA, USA) and 0.05% Pluronic F-127 (Invitrogen, Carlsbad, CA, USA) in the dark at 37 °C for 45 min in assay buffer. The cells were washed twice with assay buffer and then incubated in fresh assay buffer for 20 min. The cells were viewed on the inverted stage of a Leica TCS SP8 confocal microscope (Leica Biosystems, Wetzlar, Germany). At an excitation wavelength of 488 nm, Fluo-4 was analyzed simultaneously at 5 s intervals for BMMs or human PBMCs and at 10 s intervals for BMSCs. The increase from the basal level was determined by adding 10 μM ionomycin (Abcam, Cambridge, UK). Peak analysis was performed to obtain parameters with ImageJ2/FIJI (NIH).

Li X., Wang L., Wang H., Qin A, & Qin X. (2022). Ano5 modulates calcium signaling during bone homeostasis in gnathodiaphyseal dysplasia. NPJ Genomic Medicine, 7, 48.

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Quantifying Osteoclastogenesis with TRAP

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After BMMs were cultured with CPC or BMP2-CPC for 1 day, the culture media were replaced as follows: (1) CPC-M: CPC+BMMs+DMEM containing 30 ng/mL M-CSF; (2) CPC-OC: CPC+BMMs+DMEM containing 30 ng/mL M-CSF and 50 ng/mL RANKL (R&D, USA); (3) BMP2-CPC-M: BMP2-CPC+BMMs+DMEM containing 30 ng/mL M-CSF; and (4) BMP2-CPC-OC: BMP2-CPC+BMMs+DMEM containing 30 ng/mL M-CSF and 50 ng/mL RANKL. On day 7, the cellular TRAP activity was quantified by a TRAP activity assay kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. The TRAP activity of each sample was determined at a wavelength of 405 nm and then normalized to the corresponding total protein content.

Shen H., Zhuang Y., Zhang C., Zhang C., Yuan Y., Yu H., Si J, & Shen G. (2022). Osteoclast-Driven Osteogenesis, Bone Remodeling and Biomaterial Resorption: A New Profile of BMP2-CPC-Induced Alveolar Bone Regeneration. International Journal of Molecular Sciences, 23(20), 12204.

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Osteoclastogenesis Assay with CAR-T Cells

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MM1.S cells (20 × 10³), OPM2 cells, and H929 cells were seeded, and CAR-T cells were added at an E/T ratio of 1:5 to 5:1, respectively, for 24 hours in each well of 96-well plates. Cocultured CM were collected and added to the culture media (50%) with 50 ng/mL RANKL (R&D Systems) and 1 × 10³ RAW 264.7 cells and cultured for 7 days. The cells were then fixed in formalin and stained for TRAP using a TRAP staining kit (MilliporeSigma). TRAP⁺ cells containing 3 or more nuclei were counted as osteoclasts.
Human CD14⁺ monocytes sorted from the bone marrow of patients with MM were differentiated into osteoclasts with M-CSF (R&D Systems) and RANKL for 7 days, and CM were added to human CD14⁺ monocyte culture media (50%) for another 7 days. The cells were then fixed in formalin and stained for TRAP using a TRAP staining kit. TRAP⁺ cells containing 3 or more nuclei were counted as osteoclasts.

Sun F., Cheng Y., Chen J.R., Wanchai V., Mery D.E., Xu H., Gai D., Al Hadidi S., Schinke C., Thanendrarajan S., Zangari M., van Rhee F., Tricot G., Shaughnessy JD J.r, & Zhan F. (2024). BCMA- and CST6-specific CAR T cells lyse multiple myeloma cells and suppress murine osteolytic lesions. The Journal of Clinical Investigation, 134(1), e171396.

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Neutralizing Antibodies Characterization

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Neutralizing antibodies against CRP, MIP-1α, MCP-1, RANKL, and control IgG, and recombinant human CRP, M-CSF, RANKL, and SAP were purchased from R&D Systems (Minneapolis, MN). The p38 MAPK-specific inhibitor was purchased from Axon Medchem BV (Groningen, Netherlands). Except where specified, antibodies for western blotting analysis were purchased from Cell Signaling Technology, MA.

Yang J., Liu Z., Liu H., He J., Yang J., Lin P., Wang Q., Du J., Ma W., Yin Z., Davis E., Orlowski R.Z., Hou J, & Yi Q. (2017). C-reactive protein promotes bone destruction in human myeloma through the CD32-p38MAPK-Twist axis. Science signaling, 10(509), eaan6282.

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Osteoclastogenesis in RAW264.7 Cells

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The RAW264.7 mouse macrophage cell line, a well-known in vitro model of osteoclastogenesis, maintains the capability to differentiate into mature osteoclast-like cells.19 (link) RANKL-treated RAW264.7 cells produce high levels of osteoclast-specific markers, such as tartrate-resistant acid phosphatase (TRAP), which is commonly used to evaluate bone resorption.20
As in our previous study with RAW264.7 cell line, the RAW264.7 cell line was obtained from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco BRL, Grand Island, NY, USA) supplemented with antibiotics (100 U/mL penicillin A and 100 U/mL streptomycin) and 10% heat inactivated fetal bovine serum (FBS), and maintained at 37°C in 5% CO₂ humidified air. The cells (2 × 10⁴ cells/well) were seeded in 24-well plates and incubated to up to 70% confluence. For differentiation into osteoclasts, RAW264.7 cells were cultured in DMEM, containing 50 and 100 ng/mL RANKL (R&D Systems, Minneapolis, MN, USA) for 3 days.21 (link) To examine the effects of TBBPA on osteoclast differentiation and activity, RAW264.7 cells were treated with varying concentrations of TBBPA (10, 20, and 40 µM), in the presence of RANKL for an additional 3 days.

Park S.Y., Choi E.M., Suh K.S., Kim H.S., Chin S.O., Rhee S.Y., Kim D.Y., Oh S, & Chon S. (2019). Tetrabromobisphenol A Promotes the Osteoclastogenesis of RAW264.7 Cells Induced by Receptor Activator of NF-kappa B Ligand In Vitro. Journal of Korean Medical Science, 34(41), e267.

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