Dulbecco’s modified Eagle’s medium (DMEM), α-minimum essential medium (α-MEM), and supplements were purchased from Invitrogen Life Technologies (Carlsbad, CA). Fetal bovine serum (FBS) was obtained from HyClone Laboratories (Logan, UT). Recombinant murine sRANKL and macrophage-colony stimulating factor (M-CSF) were purchased from PeproTech (Rock Hill, NJ, USA). Tricarbonyldichlororuthenium (II) dimer (CORM-2), bilirubin, FeCl2, and ruthenium chloride (RuCl3) were purchased from Sigma-Aldrich (St Louis, MO, USA). All antibodies used in this study were purchased from Santa Cruz (Santa Cruz, CA, USA) or Cell Signaling (Beverly, MA, USA). Anti-NFATc1 monoclonal antibody was purchased from BD Biosciences Discovery Lab-ware (Bedford, MA).
Recombinant murine srankl
Manufactured by Thermo Fisher Scientific
Sourced in United States
Recombinant murine sRANKL is a soluble form of the receptor activator of nuclear factor kappa-B ligand (RANKL) protein from mouse. It functions as a key regulator of osteoclast differentiation and activation.
Automatically generated - may contain errors
Lab products found in correlation
M csf, by Thermo Fisher Scientific (3 mentions)
Leukocyte tartrate resistant acid phosphatase kit, by Merck Group (2 mentions)
α mem, by Thermo Fisher Scientific (2 mentions)
Bilirubin, by Merck Group (1 mentions)
Fecl2, by Merck Group (1 mentions)
α minimum essential medium α mem, by Thermo Fisher Scientific (1 mentions)
Tricarbonyldichlororuthenium 2 dimer corm 2, by Merck Group (1 mentions)
Macrophage colony stimulating factor m csf, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Cytiva (1 mentions)
P met, by Cell Signaling Technology (1 mentions)
Recombinant murine hgf, by Thermo Fisher Scientific (1 mentions)
P gsk3β, by Cell Signaling Technology (1 mentions)
Su11274, by Selleck Chemicals (1 mentions)
Histone 3 17168 1 ap, by Proteintech (1 mentions)
Nfatc1, by Santa Cruz Biotechnology (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
7 protocols using recombinant murine srankl
1
Osteoclastogenesis Regulation by CORM-2
2
Osteoblast and Osteoclast Differentiation Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MC3T3-E1 cells, primary calvarial osteoblasts, CRISPR-Cas9-medicated MC3T3-E1 Padi2 KO cells (#3-4 and #5-6) [8 (link)] were cultured in α-MEM with 10% fetal bovine serum (FBS) containing 100 U/mL penicillin and 100 µg/mL streptomycin in a 5% CO2 humidified atmosphere at 37 °C. To induce osteoblast differentiation, we used α-MEM growth medium supplemented with 10 mM β-glycerophosphate and 50 µg/mL ascorbic acid. The medium was changed every 2–3 days. 293 T cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) containing 100 U/mL penicillin and 100 µg/mL streptomycin. Human mesenchymal stem cells (hMSC) were purchased from STEMCELL Technologies (Vancouver, Canada) and cultured in accordance with the manufacturer’s protocol. For osteoclast differentiation, BMMs were seeded (3 × 104 cells/300 ul/ well in a 96-well plate) and differentiated into osteoclasts using 20 ng/ml M-CSF and 80 ng/ml recombinant murine sRANKL (Pepro Tech, Rocky Hill, NJ, USA) for 5 days. All cell lines used in the study were confirmed to be free of mycoplasma contamination.
3
Chondrocyte Differentiation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cell-culture reagents were provided by Gibco BRL. Recombinant murine sRANKL (#315–11) and Recombinant murine HGF (#315–23) were purchased from PeproTech. Recombinant mouse MCSF protein (#ab129146), and the following antibodies: p65 (#ab16502), TRAF6 (#ab33915), JNK (#ab124956), HGF (#ab83760), were obtained from Abcam. The following antibodies were purchased from Cell Signaling Technology: p-p65 (#3033), p-JNK (#9255), Met (#8198), p-Met (#3077), Akt (#4685), p-Akt (#4060), GSK-3β (#9315S), p-GSK-3β (#9323). NFATc1 (#sc-17834) and GAPDH (#sc-32233) antibodies were provided by Santa Cruz Biotechnology. β-actin (#60008-1-Ig) and Histone-3 (#17168-1-AP) antibodies were purchased from Proteintech Group. SU11274 was obtained from Selleck. Immunization Grade Chick type II collagen (#20012), Complete Freund’s Adjuvant (#7001), and InComplete Freund’s Adjuvant (#7002) were purchased from Chondrex.
4
Osteoclast Differentiation and Resorption Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Osteoclast differentiation and resorptive activity were studied as previously described13 (link),14 (link). Briefly, RAW264.7 cells were initially seeded in αMEM medium supplemented with recombinant murine sRANKL (Peprotech, London, UK) at day 1. Medium was refreshed after 3 days, with the addition of either 5TGM1 concentrated conditioned medium (CCM), 5TGM1 sEVs (100 µg/ml) or sRANKL. 1 day later, the osteoclast cultures were stopped, cells were fixed in 4% paraformaldehyde and stained for TRAP activity using the Leukocyte Tartrate-Resistant Acid Phosphatase kit (Sigma-Aldrich). To assess the resorptive capacity of osteoclasts, RAW264.7 cells were seeded on Osteo Assay 96-well plates (Corning, New York, USA) in osteoclast differentiation medium. The medium was refreshed every 3 days. After 12 days, a Von Kossa staining was performed to visualize non-resorbed matrix. The number of resorption pits and average pit size were quantified using ImageJ software (NIH, Bethesda, USA).
5
Osteoclast Differentiation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RAW264 cells were seeded in a 24‐well plate at the density of 3,000 cells/well. Supplemented with recombinant murine sRANKL (50 ng/ml, Peprotech, USA) and M‐CSF (10 ng/ml, Peprotech, USA), the culture medium was changed every other day. After 6 days, the Leukocyte‐Tartrate‐resistant acid phosphatase (TRAP) kit (Sigma–Aldrich; Merck KGaA) was utilised to stain the cells to detect the TRAP activity. Finally, the samples were stained with 1% aqueous Fast Green FCF for 1 min.18 Ficoll‐Paque (Salarbio) density gradient centrifugation was used to extract PBMCs. PBMCs were seeded in a 24‐well plate at the density of 1 × 106 cells/well. The culture conditions were the same as used in RAW264 cells. After 15 days, the degree of osteoclast differentiation was determined by detecting the TRAP activity of the cells.
6
Osteoclast Differentiation from BMMs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We prepared bone marrow cells and cultured BMMs according to previously reported protocols (Li et al., 2016 ), with minor modifications. Briefly, bone marrow cells were isolated from the tibia and femur of 8–12‐week‐old male mice and cultured in α‐MEM (Invitrogen) containing 10% FBS for 24 h to generate BMMs. To produce osteoclasts, BMMs were cultured in α‐MEM supplemented with recombinant murine M‐CSF (25 ng/ml, PeproTech) and recombinant murine sRANKL (50 ng/ml, PeproTech) with or without treatment with exosomes or miRNAs for 6 days. The culture medium was changed every 2 days until Day 6, and then Trap staining was performed.
7
Osteoclastogenesis Assay with HK-8 and HK-E3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Raw264.7 cells were seeded in 48-well plates at a density of 5000 cells/well in DMEM medium supplemented with recombinant murine sRANKL (Peprotech, USA) and M-CSF (Peprotech, USA) on day 2. The medium was refreshed every 3 days and treated with HK-8 (50 and 5 μM) and HK-E3 (50 and 5 μM). After 6 days, cells were stained for TRAP activity using the Leukocyte Tartrate-Resistant Acid Phosphatase kit (Sigma-Aldrich; Merck KGaA).
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!