RAW264.7 osteoclast progenitor cells (1.0 × 104 cells per well) were seeded into 48-well plates and incubated overnight. Then, the medium was exchanged, and the cells were classified into three groups: the 1) vehicle group (DMEM + PBS), 2) UHMWPE + RANKL + vehicle group (DMEM + UHMWPE + RANKL + PBS), and 3) UHMWPE + RANKL + USC-EVs group (DMEM + UHMWPE + RANKL + USC-EVs). The medium was exchanged for fresh high-glucose DMEM or DMEM + UHMWPE particles containing 100 ng·mL−1 RANKL (ProteinTech, Chicago, USA) and 300 μg·mL−1 USC-EVs or vehicle (PBS). After coculture for 7 days, the cells were stained with a commercial TRAP Kit (Sigma, USA), and the TRAP+ osteoclasts (> 3 nuclei) in each well were counted under an inverted microscope (Leica, Germany). After 4 days of induction, the original medium was collected and used for ELISAs.
Rankl
Manufactured by Proteintech
Sourced in United States, China
RANKL is a recombinant protein produced by Proteintech. It is a key regulator of osteoclast differentiation and activation, playing a crucial role in bone remodeling.
Automatically generated - may contain errors
Lab products found in correlation
Nfatc1, by Santa Cruz Biotechnology (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Inverted microscope, by Leica (1 mentions)
Trap kit, by Merck Group (1 mentions)
Runx2, by Affinity Biosciences (1 mentions)
Hrp conjugated goat anti rabbit secondary antibody, by Abcam (1 mentions)
Imagequant 350, by GE Healthcare (1 mentions)
Pparα, by Affinity Biosciences (1 mentions)
Hrp conjugated goat anti mouse secondary antibody, by Abcam (1 mentions)
Serca2, by Abcam (1 mentions)
Col1a1, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β catenin, by Proteintech (1 mentions)
β actin, by Proteintech (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
P38 mapk, by Proteintech (1 mentions)
Cathepsin k, by Proteintech (1 mentions)
Nfatc1, by Proteintech (1 mentions)
β actin, by Merck Group (1 mentions)
Tnf α, by Proteintech (1 mentions)
10 protocols using rankl
1
Osteoclast Differentiation Assay with USC-EVs
2
Western Blot Analysis of Bone Remodeling Markers
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Total cellular proteins were extracted using the RIPA lysis buffer, and a BCA assay kit (Beyotime) was used to quantify the protein concentrations. The same amounts of proteins (20 μg) were separated by polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore). Membranes were blocked in 5% low-fat milk for 1 h, and then incubated with primary antibodies against SERCA1 (1:500; Affinity Biosciences), SERCA2 (1:5000; Abcam), SERCA3 (1:5000; Proteintech), IP3R (1:1000; Affinity Biosciences), PLCβ1 (1:1000; Proteintech), P2Y2 (1:1000; Affinity Biosciences), P2Y4 (1:1000; Affinity Biosciences) P2Y12 (1:2000; Abcam), Col1a1 (1:1000; Abcam), Runx2 (1:1000; Affinity Biosciences), Osx (1:1000; Bioss), β-catenin (1:1000; Proteintech), OPG (1:1000; Abcam), RANKL (1:1000; Proteintech), DKK1 (1:2000; Proteintech), NFATc1 (1:500; Santa Cruz), Calcr (1:1000; Proteintech), Ctsk (1:1000; Affinity Biosciences), TRAP (1:2000; Abcam), PPARα (1:1000; Affinity Biosciences), p-PPARα (1:1000; Affinity Biosciences) or β-actin (1:4000; Proteintech) overnight at 4 °C. The blots were incubated with HRP-conjugated goat-anti-rabbit secondary antibody (1:5000; Abcam) or HRP-conjugated goat-anti-mouse secondary antibody (1:5000; Abcam), and then visualized using an enhanced chemiluminescence system (Image Quant 350, GE Healthcare).
3
Molecular Mechanisms in Osteoarthritis Pathogenesis
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Three weeks after the induction of OA, mice (n = 6 for each group) were sacrificed for western blot analysis. Protein samples were isolated from proximal tibia using a mortar and pestle. Tissues were lysed in a radioimmunoprecipitation assay (RIPA) lysis buffer, containing protease inhibitors and phosphatase inhibitors (Roche Diagnostics GmbH, Mannheim, Germany). Isolated proteins were fractionated using 10% sodium dodecyl sulfate-polyacrylamide gel and electro-transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Primary antibodies specific to Wnt3a (Abcam, Cambridge, MA, USA), together with NFATc1, RANKL, TNF-α, Cathepsin K, p38 MAPK, NFκB p65 (Rel A) (Proteintech, Wuhan, China), Phosph-p38 MAPK, Phosph-NFκB (Cell Signaling, Danvers, MA, USA) and β-actin (Sigma, St Louis, MO, USA) were employed. After incubation with secondary IgG antibodies conjugated with horseradish peroxidase, signals were detected with enhanced chemiluminescence. Data were presented with reference to control intensities of β-actin24 .
4
Immunohistochemical Analysis of RANKL in OKCs
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Samples from 24 patients (2 cases were lacking the epithelial component in the IHC test) with OKCs were fixed in 4% paraformaldehyde and embedded in paraffin under the guidelines of the National Institutes of Health. The tissue samples and CFMPs used for the corollary analyses were collected from the same patient. In brief, the sections were dewaxed, rehydrated and antigen-retrieved by high pressure. Subsequently, the sections were incubated with 3% hydrogen peroxide for 20 min, goat serum for 20 min under room temperature, followed by incubation with the primary antibody [receptor activator for nuclear factor-κB ligand (RANKL), 1:200, Proteintech Group, Wuhan, China; 23408-1-AP] at 4°C overnight. For the staining, a diaminobenzidine substrate kit, and hematoxylin (both from Dako, Glostrup, Denmark) were used. For RANKL evaluation, the semi-quantitative analysis of immunohistochemical staining was performed using Image-Pro Plus 6.0 and the quantification was calculated as the mean density for each protein (IOD/area).
5
RANKL and OPG Protein Expression
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The primary antibodies used in the present study were against the following antigens: RANKL (rabbit polyclonal antibody, dilution 1 : 1000, Proteintech), OPG (rabbit polyclonal antibody, dilution 1 : 300, Abcam, Cambridge, MA, USA), and β-actin (internal control, rabbit polyclonal antibody, dilution 1 : 5000, Abcam). Proteins from the knee joint were separated using SDS-PAGE then transferred to polyvinylidene difluoride membranes. The intensity of each protein was normalized using β-actin and expressed as a proportion of the control. Analysis of Western blots was performed in accordance with a previous study [21 (link)].
6
Osteoclast Differentiation and Evaluation
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RAW 264.7 cells were seeded into 48-well-plates and cultured overnight in DMEM. Then, the cells were randomized into the following three groups with three wells per group: DMEM + PBS; DMEM + RANKL + PBS; and DMEM + RANKL + USC-Exos. The medium was replaced with fresh DMEM supplemented with 100 ng/mL RANKL (ProteinTech, USA) and 300 μg/mL USC-Exos or an equal volume of vehicle (PBS) after cell adhesion. The culture medium was replaced every other day. Eight days later, the cells were fixed in 4% PFA for 25 min and then stained using a commercial TRAP kit (Sigma-Aldrich). The numbers of TRAP+ osteoclasts were counted under a microscope (Leica). The culture medium was collected on the fourth day and evaluated by ELISA [MULTI SCIENCES (LIANKE) BIOTECH, Hangzhou, China]. The levels of osteoclast-related genes were analyzed by RT-qPCR.
7
Osteoclastogenesis Signaling Pathway Assay
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Antibodies included: ERK5 (1: 1500 for western blot), MMP-9 (1: 1500 for western blot, Abcam, USA); CTSK (1: 1500 for western blot) and TRAP (1: 1500 for western blot) and RANKL (Proteintech, USA); p-ERK5 Thr218/tyr220 (1: 1500 for western blot, Cell Signaling Technology, USA); NFATc1 (1: 1500 for western blot, Santa Cruz, CA, USA); β-actin (1: 1000 for western blot, ZSGB-BIO, China); HRP-conjugated Affinipure goat anti-mouse IgG (1: 3000), peroxidase-conjugated goat anti-rabbit IgG (1: 3000, Proteintech, USA), and XMD8-92 (Selleck, USA).
8
Osteogenesis-Related Protein Expression
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After 7 days of culture, the cells were lysed, the proteins were collected, and the total protein concentration was detected by a BCA protein assay kit. The expression levels of ALP, RUNX2, OPG and RANKL were determined by Western blot. The protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride membranes (PVDF; Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk in Tris buffered saline and 0.1% Tween-20 (TBST) at room temperature to reduce nonspecific bindings of antibodies. The blots were probed with primary antibodies overnight at 4 °C to ALP (Huabio, Hangzhou, China), RUNX2 (Proteintech, Hubei, China), OPG (Abcam, Cambridge, UK), RANKL and GAPDH (Proteintech, Hubei, China). After washing in TBST, the membrane was incubated with the corresponding secondary antibody (Absin, Shanghai, China) at 37 °C for 1 hour. We used an enhanced chemiluminescent substrate kit (Biosharp, Anhui, China) to visualize the immunoreactive proteins. ImageJ software (NIH, Bethesda, MD, USA) was used to quantitatively analyze the protein expression.
9
Western Blot Analysis of Osteogenic Markers
10
Western Blot Analysis of Exosomal and Cellular Proteins
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Exosomes (10 μg) or proteins (30–50 μg) extracted from cells were diluted 1:4 (v/v) in a loading buffer (Beyotime, China), heated at 95°C for 10 min, loaded onto 10% gels (1.5 × 10 wells; Genscript, China), electrophoresed at 180 V for 50 min, and transferred to membranes by using the iBlot (Invitrogen) 8‐min program. For all subsequent antibody‐incubation and washing steps, a rocking platform was used. After blocking with a blocking buffer (Odyssey, USA) for 60 min, the membranes were incubated with primary antibodies at 4°C overnight, washed in TBST (3 × 5 min), incubated with secondary antibodies at room temperature for 60 min, washed with TBST, and then exposed to a detection reagent (Chemiluminescent HRP Substrate, Millipore, USA) for visualization. The primary antibodies were as follows: CD9 (Cell Signaling Technology, USA, 1:1000), Alix (Cell Signaling Technology, 1:1000), Annexin V (Cell Signaling Technology, 1:1000), HSP70 (Cell Signaling Technology, 1:1000), GM130 (Cell Signaling Technology, 1:1000), COL1A1 (Cell Signaling Technology, 1:1000), GAPDH (Cell Signaling Technology, 1:1000), β‐Actin (Cell Signaling Technology, 1:1000), and RANKL (Proteintech, China, 1:3000).
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