Osteoclastic differentiation of cells was evaluated using TRAP staining with the Leukocyte Acid Phosphatase Kit (Sigma-Aldrich) and TRAP activity assay (TRAP Assay Kit; Takara, Shiga, Japan). Cultured RAW264.7 cells and rBMMs were fixed in 3.7% paraformaldehyde for 10 min, treated with 0.1% Triton X-100 in phosphate-buffered saline at room temperature for 5 min, and rinsed 3 times with deionized water. Finally, cells were incubated with 0.01% naphthol AS-MX phosphate and 0.05% fast red violet LB salt in 50 mM sodium tartrate and 90 mM sodium acetate (PH = 5.0) for 1 h at 37 °C, then rinsed 3 times with deionized water. TRAP activity was measured using the cell culture supernatant after staining.
Trap assay kit
Manufactured by Takara Bio
Sourced in Japan
The TRAP assay kit is a laboratory tool designed to detect and quantify telomerase activity in cell and tissue samples. It provides a reliable and sensitive method for measuring this important enzyme that plays a crucial role in cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Leukocyte acid phosphatase kit, by Merck Group (3 mentions)
Chloroauric acid haucl4, by Merck Group (1 mentions)
Cell culture medium, by Thermo Fisher Scientific (1 mentions)
Macrophage colony stimulating factor m csf, by Thermo Fisher Scientific (1 mentions)
Rankl, by Thermo Fisher Scientific (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Bioneer (1 mentions)
Alkaline phosphatase assay kit, by Abcam (1 mentions)
Synergy h1, by Synergy Software (1 mentions)
8 protocols using trap assay kit
1
Osteoclast Differentiation Assay
2
Osteoclast Differentiation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chloroauric acid (HAuCl4) and trisodium citrate were purchased from Sigma Aldrich. (St. Louis, MO, USA), and 30 nm SNPs were purchased from Cytodiagnostics (Burlington; ON, Canada). The cell culture medium was purchased from GIBCO (Grand Island, NY, USA). Macrophage-colony stimulating factor (M-CSF) and RANKL were purchased from Peprotech (London, UK). The TRAP assay kit used here was purchased from Takara (Shiga, Japan). EZ-Cytox was purchased from Dogen (Seoul, Korea). All primers for Cathepsin K (CTSK), c-FOS, the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), TRAP, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Bioneer (Seoul, Korea). The antibodies used in this study were purchased from Invitrogen (Carlsbad, CA, USA).
3
Serum Enzyme and CRP Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole blood was collected by cardiac puncture and serum was separated from whole blood by centrifugation, 2,200 x g at 4°C for 15 min. The TRAP enzyme activity in serum was determined colorimetrically by the TRAP Assay Kit (TaKaRa, Kusatsu, Japan). The AP enzyme activity in serum was determined colorimetrically by the Alkaline Phosphatase Assay Kit (Abcam, Cambridge, MA). The serum levels of CRP were tested by Mouse C-Reactive Protein (CRP) ELISA kit (Life Diagnostics, West Chester, PA).
4
Osteoclast TRAP Activity Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After five days of culture, osteoclasts were lysed in 0.2% triton X-100 and TRAP activity was estimated using TRAP assay kit (Takara, Shiga, Japan) as per manufacturer’s instructions. In brief, the cell lysate was mixed with assay buffer (1:1) for 60 min at 37 °C. Reaction was terminated with 0.5 N NaOH and the amount of pNP produced was measured at 405 nm using a microplate reader (Synergy H1). Concentrations of pNP were calculated using a pNP standard curve. The TRAP activity was normalized to total protein of cell lysate. TRAP is expressed as µM of pNP produced per min per µg of protein.
5
Optimal RANKL Incubation Time for Osteoclastogenesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To investigate the optimal RANKL incubation time, RAW 264.7 cells with and without 20 ng/mL RANKL treatments were incubated with CRE-SD-FREE-LIP for 1, 2, 3, 4, and 5 days. A TRAP assay was performed to detect TRAP activity. The TRAP assay kit was purchased from TaKaRa Bio Inc., Tokyo, Japan. The relative expression of CTSK was measured using the qRT–PCR procedure described by Pengjam et al. [39 (link)].
6
RANKL-Induced Osteoclastogenesis Modulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RAW 264.7 cells with RANKL (20 ng/ml) and CRE-Ter (0, 5, 10, 20 or 30 μg/ml) were cultured separately in 12 well culture vessel in DMEM containing 10% heat inactivated FBS, 2 mM glutamine, 100 U/ml penicillin G, and 100 μg/ml streptomycin sulfate for 5 days in a humidified chamber (5% CO2, 37 °C). At the end of this time period, TRAP assay was conducted using TRAP assay kit (TaKaRa, Bio, Inc, Tokyo, Japan) according to the manufacturer's instructions. TRAP-positive multinucleated cells containing dark orange cells were monitored under a light microscope (Nikon, model # MFA20100, Japan).
7
Osteoclast Differentiation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Osteoclastic differentiation of BMMs was evaluated by TRAP staining using the Leukocyte Acid Phosphatase Kit (Sigma-Aldrich, St. Louis, MO, USA) and TRAP activity assay (TRAP Assay Kit; Takara, Shiga, Japan). Cultured cells were fixed in 3.7% paraformaldehyde for 10 minutes, treated with 0.1%Triton X-100 in PBS at room temperature for 5 minutes, and rinsed three times with deionized water. Finally, cells were incubated with 0.01% naphthol AS-MX phosphate and 0.05% fast red violet LB salt in 50mM of sodium tartrate and 90mM of sodium acetate (pH 5.0) for 1 hour at 37 ℃ and rinsed 3 times with deionized water. TRAP activity was measured by using the cell culture supernatant generated after staining.
8
Osteoclast Characterization by TRAP Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TRAP staining was performed using an acid phosphatase leukocyte kit (Sigma-Aldrich) in osteoclasts fixed with 4% paraformaldehyde. TRAP-positive multinuclear cells containing more than three nuclei were scored as osteoclasts and examined using an Olympus microscope (40×) (Model IX71, Olympus, Center Valley, PA, USA). TRAP activity was determined using a TRAP assay kit (Takara Bio, Shiga, Japan). Briefly, 50 μL of cell extract and 50 μL of p-nitro-phenyl phosphate ( pNPP) were mixed with a sodium tartrate solution and incubated at 37 °C for 25 min. The reaction was stopped with 0.5 N NaOH and the absorbance was measured at 405 nm. A solution containing acid phosphatase and pNPP, but not the sodium tartrate solution, was used as a control.
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!