BMMs were cultured with density of 10000 cells/well in a 96-well plate. RANKL (50 ng/ml) without or with Vericiguat (0, 100 μM, 500 nM, 1 μM, 2 μM, 4 μM, and 8 μM) was added at the second day for 6 d. Recombinant mouse-derived M-CSF and RANKL were purchased from Novoprotein Scientific Inc. (Pudong New District, China). The identification of OC was evaluated by TRAP staining using a tartrate resistant acid phosphatase staining kit (Sigma-Aldrich Institute of Biotechnology, St. Louis, MO, USA). TRAP-positive cells were observed and imaged using light microscopy (Nikon, Tokyo, Japan).
Rankl
Manufactured by Novoprotein
Sourced in China
RANKL is a recombinant protein produced by Novoprotein. It is a member of the tumor necrosis factor (TNF) superfamily and plays a crucial role in the regulation of bone metabolism and the immune system.
Automatically generated - may contain errors
Lab products found in correlation
M csf, by Novoprotein (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Double antibiotics, by Thermo Fisher Scientific (1 mentions)
Dmem f12 1 1 medium, by Thermo Fisher Scientific (1 mentions)
Raw264.7 cells, by Shanghai Zhong Qiao Xin Zhou Biotechnology (1 mentions)
Bm mscs, by Shanghai Zhong Qiao Xin Zhou Biotechnology (1 mentions)
Trap kit, by Merck Group (1 mentions)
Lactate dehydrogenase assay kit, by Solarbio (1 mentions)
Tartrate resistant acid phosphatase stain kit, by Solarbio (1 mentions)
Actin tracker red rhodamine, by Beyotime (1 mentions)
Complete freund s adjuvant cfa, by BD (1 mentions)
Foetal bovine serum (fbs), by Corning (1 mentions)
Rabbit anti mouse igg, by Abcam (1 mentions)
Raw264, by American Type Culture Collection (1 mentions)
α mem, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Solarbio (1 mentions)
Interleukin 4 (il 4), by Sino Biological (1 mentions)
Nfatc1 66963 1 ig, by Proteintech (1 mentions)
Trap staining kit, by Merck Group (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
9 protocols using rankl
1
Osteoclast Differentiation with Vericiguat
2
Murine Primary BMMSCs and RAW264.7 Osteoclasts
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The murine primary BMMSCs were obtained from Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (#ZQ0465, Shanghai, China). BMMSCs were cultured in DMEM/F12 (1:1) medium (#11320033, Gibco, US) containing 10% fetal bovine serum (FBS) and 1% double antibiotics (penicillin/streptomycin mix) (#10378016, Gibco, USA). The BMMSCs were maintained in a humidified atmosphere of 5% CO2 at 37 °C. RAW264.7 cells were purchased from Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (#ZQ0098, Shanghai, China), cultured with DMEM supplemented with 10% FBS and 1% antibiotics, and incubated in a humidified atmosphere of 5% CO2 at 37 °C. For osteoclast differentiation induction, RAW264.7 cells were treated with 50 ng/mL RANKL (#CJ94, Novoprotein, Beijing, China).
3
Osteoclast Formation Assay with PTH and NE
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The BMMs were cultured in 48-well plates in α-MEM (HyClone, Logan, USA) supplemented with 50 ng/mL of receptor activator for nuclear factor κB ligand (RANKL; Novoprotein, Shanghai, China) in the presence of vehicle (PBS), PTH (100 ng/mL), PTH + NE (10 μmol/L), PTH (100 ng/mL) + NE (10 μmol/L) + propranolol (1 μmol/L) or conditioned media from BMSCs treated with vehicle iPTH, iPTH + NE or iPTH + NE + propranolol as described above. Osteoclast formation was identified using a commercial TRAP kit (Sigma‒Aldrich, USA) according to the manufacturer’s instructions. The areas of TRAP+ (red) osteoclasts were measured by ImageJ 1.8.0 software.
4
Tan IIA-Induced Osteoclastogenesis Proteomics
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Tan IIA was purchased from Beijing Bethealth People Biomedical Technology (Beijing, China; purity ≥ 98%). Complete Freund's adjuvant (CFA) was purchased from Becton, Dickinson and Company (USA). Foetal bovine serum was obtained from Corning (USA), and penicillin/streptomycin was obtained from Thermo Fisher (USA). RANKL was obtained from Novoprotein (Suzhou). Cell fixative was obtained from Biorigin (Beijing). The Tartrate-Resistant Acid Phosphatase (TRAP) Stain Kit was obtained from Solarbio (Beijing). Actin-Tracker Red-Rhodamine was obtained from Beyotime Biotechnology (Beijing). A lactate dehydrogenase assay kit was obtained from Solarbio (Beijing). The NAD/NADH assay kit was obtained from BioXcellence (Beijing). Rabbit anti-mouse IgG was obtained from Abcam (Beijing). The desthiobiotin iodoacetamide (DBIA) probe was obtained from ChomiX Biotech (Nanjing). The following click chemistry reaction and LC‒MS/MS reagents were used in this study: TBTA (1770049), TCEP (C4706), rhodamine-N3 (83689) and CuSO4 (C1297) were purchased from Sigma (USA); TMT 10plex™ reagent (A34808), high-capacity neutravidin agarose resin (A53031) and sequencing grade modified trypsin (90057) were purchased from Thermo Fisher (USA).
5
Osteoclast Differentiation of RAW 264.7 Cells
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RAW 264.7 and HEK-293T cells, purchased from the Cell Bank of Chinese Academy of Sciences, were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. All cell lines were cultured at 37°C with 5% CO2.
In order to induce osteoclast differentiation, RAW 264.7 cells were incubated with RANKL (100 ng/mL; Novoprotein Scientific, Shanghai, China) for 5 days.
In order to induce osteoclast differentiation, RAW 264.7 cells were incubated with RANKL (100 ng/mL; Novoprotein Scientific, Shanghai, China) for 5 days.
6
Murine Macrophage Polarization and Osteoclastogenesis
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Murine macrophage lines RAW264.7 were purchased from American Type Culture Collection (ATCC, USA) and maintained in α-MEM (Sigma, USA) supplemented with 10% fetal bovine serum (FBS, Lonsera, Uruguay) and 1% antibiotics (penicillin 100 U/ml, streptomycin 100 μg/ml ) providing atmosphere of 37℃ and 5% CO2. In order to research macrophages polarization, cells were treated with different concentrations of AGEs (Bioss, China), 1μg/ml LPS (Solarbio, China) and 20ng/ml IL-4 (Sino Biological, China). To induce macrophages to differentiate into osteoclasts, α-MEM containing 50ng/ml RANKL (Novoprotein, China) and 30ng/ml M-CSF (Novoprotein, China) was used to culture cells for 6 days. The rst 3 days of osteoclasts differentiation were considered to be the early stage, and the last 3 days were considered to be the late stage. Three repeat holes were set for each sample.
7
Osteoclastogenesis Regulation Pathway
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M-CSF (#CB34) and RANKL (#CR06) were both acquired from Novoprotein (Shanghai, China). FBS (#E01021) was purchased from Evacell (Hong Kong, China). Alpha-modified MEM (α-MEM) and penicillin‒streptomycin were obtained from HyClone (USA). Rabbit polyclonal antibodies against LC3 (#14600-1-AP), ATG5 (#10181-2-AP), Beclin1 (#11306-1-AP), P62 (#18420-1-AP), CTSK (#11239-1-AP), and NFATc-1 (#66963-1-Ig) were acquired from Proteintech Group (Wuhan, China).
8
Osteoclast Differentiation of RAW264.7 Cells
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α-minimum essential medium (MEM) (Gibco, USA) comprising 1% penicillin-streptomycin, 10% FBS, and 50 ng/mL receptor activator of nuclear factor kappa-Β ligand (RANKL) (Novoprotein, China) was used to induce RAW264.7 cells for 5 days. The medium was changed every 2 days, and the TRAP staining kit (Sigma-Aldrich, USA) was used to stain the TRAP in the osteoclasts after the formation of mature osteoclasts. An osteoclast having three or more nuclei was considered mature if it was TRAP-positive. Inverted microscopy was used to measure and score the number and size of the mature osteoclasts.
9
Osteoclastogenesis and Bone Resorption Assay
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For the osteoclastogenesis assay, BMCs were isolated from the mouse monocyte depletion model as mentioned above. Cells were cultured for 5–6 days in α-modified Eagle’s medium (α-MEM, HyClone, Logan, Utah, USA) containing 10% FBS, 1% penicillin–streptomycin (Invitrogen), receptor activator of nuclear factor kappa B ligand (RANKL) (25 ng/ml, Novoprotein, Suzhou, China), and macrophage colony-stimulating factor (MCSF) (40 ng/ml, Novoprotein) in an incubator under 5% CO2 at 37 °C35 (link),38 (link). Cells were fixed and stained for TRAP. Digital images were acquired using a light microscope.
For the bone resorption assay, cells were plated on Corning Osteo Assay Surface 24-well plates (Corning, Corning, NY, USA) and cultured in 40 ng/ml MCSF and 25 ng/ml RANKL for 14 days. Cells were removed using an ultrasonic cleaner, and resorption pits were captured using a light microscope (Leica).
For the bone resorption assay, cells were plated on Corning Osteo Assay Surface 24-well plates (Corning, Corning, NY, USA) and cultured in 40 ng/ml MCSF and 25 ng/ml RANKL for 14 days. Cells were removed using an ultrasonic cleaner, and resorption pits were captured using a light microscope (Leica).
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