PBMCs, as previously described, were harvested and cultured with M-CSF (50 ng/mL) and RANKL (R&D Systems, 100 ng/mL) for 7–10 days. The culture medium was exchanged with fresh medium every 2 days, and osteoclast formation was evaluated. TRAP staining was performed with the TRAP assay kit (Sigma Aldrich) according the manufacturer. Cells were fixed with 3.7% formaldehyde for 30 sec and incubated for 1 hr at 37 °C, with protection from light in a mixture of fast Garnet GBC, sodium nitrite, naphthol AS-BI phosphoric acid, acetate, and the tartrate of the leukocyte acid phosphatase.
Trap assay kit
Manufactured by Merck Group
Sourced in United States
The TRAP assay kit is a laboratory tool used to measure the activity of tartrate-resistant acid phosphatase (TRAP), an enzyme associated with osteoclast function. The kit provides a standardized method for quantifying TRAP activity in samples, which can be useful for researchers studying bone metabolism and related diseases.
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Lab products found in correlation
Rankl, by Thermo Fisher Scientific (2 mentions)
Trap staining kit, by Merck Group (2 mentions)
M csf, by R&D Systems (1 mentions)
Rankl, by R&D Systems (1 mentions)
Anti phospho jnk antibody, by Cell Signaling Technology (1 mentions)
Anti phospho p38 antibody, by Cell Signaling Technology (1 mentions)
Anti mmp9 antibody, by Abcam (1 mentions)
Anti phospho erk antibody, by Cell Signaling Technology (1 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Anti c fos antibody, by Santa Cruz Biotechnology (1 mentions)
Osteo assay surface multiple well plate, by Corning (1 mentions)
Anti traf6 antibody, by Santa Cruz Biotechnology (1 mentions)
Anti nfatc1 antibody, by BD (1 mentions)
Anti total erk antibody, by Cell Signaling Technology (1 mentions)
Anti total p 38 antibody, by Cell Signaling Technology (1 mentions)
Dulbecco s phosphate buffered saline, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Eclipse 90i, by Nikon (1 mentions)
Evos fl auto microscope, by Thermo Fisher Scientific (1 mentions)
Eclipse te2000 s microscope, by Nikon (1 mentions)
15 protocols using trap assay kit
1
Osteoclast Differentiation from PBMCs
2
Osteoclast Differentiation Assay
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TRAP activity is a particular biochemical activity marker of osteoclasts, which was determined to confirm osteoclast differentiation. Briefly, RAW264.7 cells with or without transfection were seeded in plates with 48 wells (1 × 103 cells/well). After 24 h of culturing, we cultured cells with 100 ng/mL RANKL in presence or absence of 2 μM AM1241 or 200 nM AM630 for 5 days. Then, after washing with PBS for three times, we fixed cells in 4% paraformaldehyde for 10 min. Then we stained them for TRAP through a commercial kit (Sigma-Aldrich). We classified TRAP-positive multinucleated cells (TRAP + MNCs) containing ≥ 3 nuclei for osteoclast, counted and captured them by a microscope (Olympus, Tokyo, Japan). For TRAP activity measure, the culture medium was collected, and measured with a TRAP assay kit (Sigma-Aldrich), we detected optical density at 405 nm by a microplate reader.
3
Osteoclastogenesis Regulation by RANKL
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RANKL was purchased from Peprotech (London, UK). Alpha-minimum essential media (α-MEM), fetal bovine serum (FBS), penicillin/streptomycin (P/S) and Dulbecco’s phosphate buffered saline (DPBS) were obtained from Gibco (Gaithersburg, NY, USA). TRAP assay kit was obtained from Sigma Aldrich (Saint Louis, MO, USA). Osteo assay surface multiple well plates were obtained from Corning, Inc. (New York, NY, USA). Anti-c-Fos antibody, anti-TRAF6 antibody and anti-β-actin antibody were obtained from Santa Cruz (CA, USA). Anti-NFATc1 antibody was purchased from BD Pharmingen (San Diego, CA, USA). Anti-MMP-9 antibody and anti-CTK antibody were purchased from Abcam (Cambridge, MA, USA). Anti-total-ERK antibody, anti-phospho ERK antibody, Anti-total-JNK antibody, anti-phospho JNK antibody, Anti-total-p38 antibody and anti-phospho p38 antibody were purchased from Cell signaling (Beverly, MA, USA). Anti-NFATc1 antibody was purchased from BD Pharmingen (San Diego, CA, USA).PCR primers were obtained from Genotech (Daejeon, Korea). All of the chemicals used in the experiments were of analytical grade or complied with the level required for cell culture.
4
Quantifying Osteoclast Differentiation from FL Cells
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To quantify differentiation and fusion of FL-derived OCs (FL-OCs) from RhoE+/+, RhoE+/gt, and RhoEgt/gt mice (Mocholi et al., 2011 (link)), the same number of precursors (2 × 104 cells/well) from E15.5 littermates was seeded in 96-well, culture-treated plates and on bones slices of the same size. They were then fixed at several time points (days 5, 6, and 8 postseeding) with 4% (wt/vol) paraformaldehyde in PBS for 15 min at room temperature and then stained at 37°C using a TRAP assay kit (Sigma-Aldrich) according to manufacturer's instructions. Stained cells with three or more nuclei were counted as OCs.
5
Quantifying Osteoblasts and Osteoclasts
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After μCT scans, right tibiae were decalcified and processed for paraffin sections. Five-μm longitudinal sections were stained either by hematoxylin and eosin (H&E) for counting the number of cuboidal and plump bone lining osteoblasts, or by tartrate-resistant acid phosphatase (TRAP) assay kit (Sigma-Aldrich, St. Louis, MO, USA) for counting the number of TRAP-positive multinucleated osteoclasts within the secondary spongiosa. All images were captured by Nikon Eclipse 90i and quantified using Bioquant Osteo Software (Bioquant Image Analysis, Nashville, TN, USA).
6
Taxifolin Inhibits Osteoclast Formation
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To examine the effects of taxifolin on osteoclast formation in cultured BMMCs and RAW264.7 cells, besides RANKL (50 ng/ml) and/or M-CSF (25 ng/ml), cells were treated with taxifolin as indicated concentrations for 4 days (Satue et al., 2013 (link)). Osteoclasts were identified by tartrate-resistant acid phosphatase (TRAP) staining kit (Sigma-Aldrich) according to the manufacturer’s protocol. TRAP-positive multinucleated cells with three or more nuclei were identified as osteoclasts (Asagiri and Takayanagi, 2007 (link)). Cell images were taken using a digital camera attached to an EVOS FL Auto microscope (Life Technologies, United States). TRAP enzyme activity in cultured medium collected from BMMCs was measured with a TRAP Assay Kit (Sigma-Aldrich, Shanghai, China) following the manufacturer’s instructions. TRAP enzyme activity was quantified using a Synergy fluorescence plate reader at 405 nm on a colorimetric plate reader.
7
Osteoclast Quantification and TRAP Activity
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Four days after treatment, tartrate resistant acid phosphatase (TRAP) staining was performed on cultured RAW264.7 cells and BMMCs with a TRAP staining kit (Sigma–Aldrich, Shanghai, China) according to the manufacturer’s protocol. TRAP-positive cells with three or more nuclei were counted as osteoclasts (Asagiri and Takayanagi, 2007 (link)). Cell images were taken using a digital camera attached to a Nikon ECLIPSE TE2000-S microscope (Nikon, Japan). TRAP enzyme activity was measured with a TRAP Assay Kit (Sigma–Aldrich, Shanghai, China) following the manufacturer’s instructions. Briefly, culture medium was collected from osteoclasts formed by BMMCs. TRAP enzyme activity was measured with a Synergy fluorescence plate reader at 405 nm on a colorimetric plate reader.
8
Histological Analysis of Bone Samples
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After micro-CT analysis, samples were embedded in paraffin, prepared into 5-μm sections, and subjected to histological and immunohistochemical examinations. First, sections were stained with Masson’s trichrome to evaluate bone collagen expression. Next, a rabbit polyclonal antibody against osteocalcin (OCN; 1:500 in PBS; ab93876, Abcam) was used to detect expression of OCN (a marker protein of late-stage osteoblast differentiation) in accordance with the manufacturer’s instructions. Subsequently, the expression of osteoclasts in ossified samples was assessed using a tartrate-resistant acid phosphatase staining (TRAP) assay kit (Sigma Aldrich), in accordance with a previous report (Sun et al., 2020 (link)).
9
Osteoclastogenesis Induction from Bone Marrow Macrophages
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BMM cells were isolated from long bone of 1-month-old WT mice and plated into 24-well culture plates at a density of 4 × 105 cells per well and cultured in αMEM with 10% FBS. Cells were treated with 20 ng·mL−1 macrophage colony-stimulating factor (M-CSF) for 3 days, and then switched to the medium with 10 ng·mL−1 M-CSF and 50 ng·mL−1 RANKL for 7 days. To test Axin1Osx CM, BMM cells were treated with CM collected from cultured calvarial osteoblasts isolated from Axin1Osx KO mice and Cre-negative controls for 7 days. The culture medium was changed every 3 days. TRAP staining was performed using a TRAP assay kit (Sigma, St. Louis, MO, USA).
10
Osteoclastogenesis Inhibition via Rutin
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Minimum essential Eagle's medium, α-modification (α-MEM), fetal bovine serum (FBS) and penicillin/streptomycin (P/S) were supplied by Gibco; Thermo Fisher Scientific, Inc. Dulbecco's modified Eagle's medium (DMEM) was procured from Welgene, Inc. Alizarin Red S was obtained from Duksan Co., Ltd. RANKL was purchased from Peprotech, Inc. Rutin, dimethylsulfoxide (DMSO) and the TRAP assay kit were purchased from Sigma-Aldrich; Merck KGaA. Osteo strip well plates were purchased from Corning Inc. Anti-RUNX2, anti-BMP-2, anti-Ctsk and anti-MMP-9 antibodies were purchased from Abcam. Anti-phosphorylated (p)-extracellular signal-regulated kinase 1/2 (ERK1/2), anti-ERK1/2, anti-p-c-Jun N-terminal kinase (JNK), anti-JNK, anti-p-p38, anti-p38, anti-NF-κB and anti-p-NF-κB antibodies were supplied by Cell Signaling Technology, Inc. Anti-NFATc1 was procured from BD Biosciences, and anti-c-Fos, anti-actin and anti-lamin B antibodies were purchased from Santa Cruz Biotechnology, Inc. Secondary antibodies were procured from Jackson ImmunoResearch Laboratories, Inc. The reverse transcriptase kit and SYBR-Green solution was supplied by Invitrogen; Thermo Fisher Scientific, Inc. Taq polymerase was purchased from Kapa Biosystems; Roche Diagnostics. PCR primers were purchased from Genotech Corp. All of the chemicals used in the experiments were analytical grade for cell culture.
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