We added sRANKL (PeproTech, Inc., Rocky Hill, NJ, USA) to cancer cells to final concentration of 10 µg/ml for 0, 5, 10, 30 or 60 min. We added 10 µM PP2 (Sigma-Aldrich St. Louis, MO, USA) or Nystatin (50 µg/ml; cat. no. N3503; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany and/or its affiliates) 1 h prior to sRANKL. To detect the lipid raft aggregation, we used CTXB (1 mg/ml; cat. no. SAE0069-500UG; Sigma-Aldrich; Merck KGaA).
Srankl
Manufactured by Merck Group
Sourced in United States, Germany
SRANKL is a laboratory reagent manufactured by Merck Group. It is a recombinant soluble receptor activator of nuclear factor kappa-B ligand (sRANKL) protein. sRANKL is a key regulator of osteoclast differentiation and bone resorption.
Automatically generated - may contain errors
Lab products found in correlation
Srankl, by Thermo Fisher Scientific (2 mentions)
Nystatin, by Merck Group (1 mentions)
α minimum essential medium, by Thermo Fisher Scientific (1 mentions)
Ficoll, by GE Healthcare (1 mentions)
Srankl, by Oriental Yeast (1 mentions)
Donepezil, by Merck Group (1 mentions)
Kenpaullone, by Merck Group (1 mentions)
Olomoucine, by Merck Group (1 mentions)
M csf, by Merck Group (1 mentions)
Soluble rankl srankl, by Thermo Fisher Scientific (1 mentions)
Srankl, by Fujifilm (1 mentions)
M csf, by Fujifilm (1 mentions)
Roscovitine, by Merck Group (1 mentions)
Ddy mice, by Japan SLC (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Macrophage colony stimulating factor m csf, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
17β estradiol e2, by Merck Group (1 mentions)
6 protocols using srankl
1
Lipid Raft Aggregation in Cancer Cells
2
Monocyte Differentiation into Osteoclasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells (PBMCs) were obtained from the blood of healthy donors using Ficoll (GE Healthcare, Buckinghamshire, UK), while CD14 + cells were purified as previously described (7) . Flow cytometry analysis using phycoerythrin-conjugated antihuman anti-CD14 monoclonal antibody (Miletenyi Biotec, Bergisch Gladbach, Germany) showed that the purity of CD14 + monocytes was more than 98% in each experiment. Purified CD14 + monocytes were seeded in 96well plates (5×10 4 cells/well) for tartrate-resistant acid phosphatase (TRAP) staining and in 24-well plates (2.5×10 5 cells/well) for quantitative real-time PCR. Cells were cultured in α-minimum essential medium (Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and incubated with macrophage-colony stimulating factor (M-CSF) (25 ng/mL) (Merck Millipore, Darmstadt, Germany) and soluble receptor activator of NF-κB ligand (RANKL) (sRANKL; 40 ng/mL; Merck Millipore) with or without metformin (1 μM) for 7 days, and then cells were subjected to TRAP staining or quantitative real-time PCR analysis.
3
Donepezil Prevents RANKL-Induced Bone Loss
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All mice (C57BL/6) were obtained from Tokyo Laboratory Animals Science, and were kept on a normal laboratory chow in an environmentally controlled clean room at the Department of Oral and Maxillofacial Surgery, Saitama Medical University. The experiments were conducted according to the institutional guidelines for ethical animal experiments. To obtain the mouse model of RANKL-induced bone loss, soluble RANKL (sRANKL, 1 mg/kg, Oriental Yeast, Japan) was injected intraperitoneally at 24 h intervals for 3 days into male mice (9-week-old) as previously described [18] (link). To evaluate the prophylactic effect of donepezil, donepezil (2 mg/kg, Sigma-Aldrich, USA) was injected before each injection of sRANKL three times at 24 h intervals intraperitoneally. This dose of donepezil was selected on the basis of previous studies [19] (link)
[20] (link)
[21] (link). The mice were killed 90 min after the last injection. Blood samples were collected at the time of sacrifice. Six mice were used in each group.
[20] (link)
[21] (link). The mice were killed 90 min after the last injection. Blood samples were collected at the time of sacrifice. Six mice were used in each group.
4
Osteoclast Differentiation from Murine Precursors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the osteoclastogenic culture, RAW264.7 and pNFAT/Luc-RAW cells were cultured in α-MEM medium containing 10% fetal bovine serum (FBS), 100 ng/ml soluble RANKL (sRANKL, Peprotech and Oriental Yeast), 2 mM l -glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin [10] (link). For osteoclast formation by using primary pre-osteoclast cultures, murine bone marrow cells were obtained from femurs and tibiae of 7-week-old ddY mice (Japan SLC, Inc.). 4.0×105 cells were cultured in α-MEM medium containing 10% fetal bovine serum (FBS), 100 ng/ml sRANKL, 10 ng/ml M-CSF (Wako, JPN), 2 mM l -glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin in 24-well plates. Kenpaullone (Sigma), olomoucine (Sigma), roscovitine (Sigma) are used at a final concentration of 50 nM. The culture medium in each well was replaced by fresh medium containing M-CSF and sRANKL every 2 days. TRAP staining was performed after 5 days of the induction.
5
Isolation and Identification of Kushennol F and Sohoravanone G
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The compounds of Kushennol F (KF) and Sohoravanone G (SG) were isolated and identified as previously described [21 (link)]. Cell culture medium including Alpha-modified Minimum Essential Medium Eagle (α-MEM), Phenol Red Free α-MEM, Dulbecco’s Modified Eagle’s Medium (DMEM), and cell culture serum including Fetal Bovine Serum (FBS) and Dextran-charcoal-stripped Fetal Bovine Serum (sFBS) were purchased from Gibco (Gibco, Gaithersburg, MD, USA). Penicillin and streptomycin were purchased from Invitrogen (Invitrogen, Carlsbad, CA, USA). 17β-estradiol (E2), receptor activator of nuclear factor κB (NF-κB) ligand (sRANKL), and Macrophage Colony-Stimulating Factor (M-CSF) were purchased from Sigma Aldrich (Sigma, St. Louis, MO, USA).
6
Osteoclastogenesis from RAW264 and MSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RAW264 cells were seeded in 24-well plates (Falcon) at 5 × 10 3 cells/well. On the following day (day 0), RANK Ligand, Soluble, Murine, Recombinant (sRANKL; Pepro Tech, Rocky Hill, CT, USA) was added to α-MEM (Sigma Aldrich) at 100 ng/mL. The medium was replaced with medium containing sRANKL every 2nd day. On day 8, TRAP staining was performed using a TRAP/ALP staining kit (Wako, Tokyo, Japan) to confirm differentiation into osteoclasts, and cells were observed with a DMi1 microscope (Leica, Wetzlar, Germany).
Next, MSCs were seeded into 6-well plates (Falcon) at 1 × 10 4 cells/well, and RAW264 cells were seeded at In other 6-well plates (Falcon), RAW264 cells were cultured alone at 5 × 10 3 cells/well. On the following day (day 0), the culture medium was replaced with medium containing sRANKL (Pepro Tech) (sRANKL was added to both RAW264 only cultures and RAW264/MSC co-cultures), and the medium was replaced with fresh medium every 2nd day.
From day 0 (at the start of sRANKL addition), using the number of actin ring-forming cells with five or more nuclei to represent the number of mature osteoclasts, osteoclast counts among RAW264 cells cultured alone and in RAW264 cell/MSC co-cultures were compared.
Next, MSCs were seeded into 6-well plates (Falcon) at 1 × 10 4 cells/well, and RAW264 cells were seeded at In other 6-well plates (Falcon), RAW264 cells were cultured alone at 5 × 10 3 cells/well. On the following day (day 0), the culture medium was replaced with medium containing sRANKL (Pepro Tech) (sRANKL was added to both RAW264 only cultures and RAW264/MSC co-cultures), and the medium was replaced with fresh medium every 2nd day.
From day 0 (at the start of sRANKL addition), using the number of actin ring-forming cells with five or more nuclei to represent the number of mature osteoclasts, osteoclast counts among RAW264 cells cultured alone and in RAW264 cell/MSC co-cultures were compared.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!