ALP activity of ADSCs groups and ADSCs sheet groups were determined using TRACP & ALP assay kit (MK301, TaKaRa Bio, Japan) according to the manufacturer’s instruction. The check points were set at Day 1, 3, 7, 10, 14 and 21.
Tracp alp assay kit
Manufactured by Takara Bio
Sourced in Japan
The TRACP & ALP Assay Kit is a laboratory tool designed to quantitatively measure the activities of Tartrate-Resistant Acid Phosphatase (TRACP) and Alkaline Phosphatase (ALP) in biological samples. The kit provides a straightforward protocol and reagents to enable the simultaneous assessment of these two important enzymatic markers.
Automatically generated - may contain errors
Lab products found in correlation
Acid phosphatase kit, by Merck Group (4 mentions)
Multiskan sky, by Thermo Fisher Scientific (3 mentions)
Bcip nbt, by Merck Group (2 mentions)
Ctgf or ccn3, by Thermo Fisher Scientific (2 mentions)
Osteoassay bone plates, by Lonza (2 mentions)
Microvue bone helical peptide eia assay, by Quidel (2 mentions)
Trap alp staining kit, by Fujifilm (2 mentions)
Spectramax 50s, by Molecular Devices (1 mentions)
Calcium e ha test wako, by Fujifilm (1 mentions)
Neuraminidase, by Roche (1 mentions)
Vector red alkaline phosphatase substrate kit, by Vector Laboratories (1 mentions)
Calcium nitrate tetrahydrate ca no3 2 4h2o, by Merck Group (1 mentions)
Ammonium phosphate dibasic nh4 2hpo4, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Abi prism 7500 sequence detector, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Mem alpha medium, by Thermo Fisher Scientific (1 mentions)
48 well cell culture plate, by Corning (1 mentions)
39 protocols using tracp alp assay kit
1
Measuring ALP Activity in ADSCs
2
Measurement of ALP Activity
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The enzymatic activity of ALP was determined using a TRACP & ALP Assay Kit (TaKaRa Bio, Shiga, Japan) in accordance with the manufacturer’s protocols. Briefly, the cells were washed with saline and solubilized with 1% NP-40. The cell extracts were incubated in solution containing p-nitrophenyl phosphate at 37 °C. The reaction was stopped by the addition of 0.5 N NaOH, and the absorbance at 405 nm was measured with a microplate reader SpectraMax 50S (Molecular Devices Japan, Tokyo, Japan). A standard curve was constructed using calf intestinal ALP (TaKaRa Bio), and the ALP activity in the samples was calculated. One unit (U) of ALP activity represents the hydrolysis of 1 μmol p-nitrophenyl phosphate per minute at 37 °C.
3
Alkaline Phosphatase Activity Assay
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ALP activity was evaluated using a TRACP & ALP assay kit (TakaRa, Shiga, Japan). MC3T3-E1 cells were cultured on a 48-well plate with a cell density of 1.5 × 104 cells mL−1 for 7 and 14 days. After culture, the medium was removed and the plate was rinsed with saline solution. The extraction and ALP buffer solutions adding p-Nitrophenyl Phosphate (pNPP) solution reacted with the cells in a 37 °C incubator for 1 h. 100 μL of reaction solution was placed on a 96-well plate, and the absorbance was measured at 405 nm using an ELISA reader.
4
Neuraminidase Effects on Osteoclast Activity
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Example 2
Neuraminidase (0.1 U/mouse) (manufactured by Roche Ltd., derived from Arthrobacter ureafaciens) or PBS was intravenously administered to 8-12 week old male C57BL/6J strain wild type (WT) and DCIR knockout mice (Dcir−/−) five times every two days. 24 hours after the last administration, whole blood was collected by cardiac puncture under anesthesia. Next, serum was prepared, and the calcium ion concentration of the serum was measured using Calcium E-HA Test Wako (manufactured by FUJIFILM Wako Pure Chemical Corporation). The serum TRAP activity, which is a marker for osteoclasts, was measured by absorbance at a wavelength of 405 nm using TRACP & ALP Assay Kit (manufactured by Takara Bio Inc.) according to the instruction manual of the kit.
Serum calcium concentration and TRAP activity in wild type mice were significantly reduced (p<0.05) by administration of Neuraminidase (
5
Screening Alkaline Phosphatase Activity
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iCSCL-10A cells (5×103 cells/well) were plated in 100 ml/well in 96-well plates. After 24 hrs, 1 mM of each compound was added to the appropriate wells. Alkaline Phosphatase (ALP) activity was measured after 48 hrs using the TRACP & ALP Assay Kit (TaKaRa, Shiga, Japan). For microscopic examination, cells were stained using the VECTOR Red Alkaline Phosphatase Substrate Kit (VECTOR Laboratories, Burlingame, CA) according to the manufacturer's protocol.
6
Synthesis and Characterization of GelMA Hydrogels with BCP-NPs
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All materials used in the synthesis of GelMA (Type A gelatin, Methacrylic anhydride (MA), dialysis tubing, high-retention seamless cellulose tubing (12–14 kDA MWCO, 40 mm diameter), Dulbecco’s phosphate-buffered saline (DPBS) Dialysis tubing closures), fabrication of hydrogel (Triethanolamine (TEA), Eosin Y disodium salt, N-Vinylcaprolactam (VC)), fabrication of biphasic calcium phosphate nanoparticles (BCP-NP) (calcium nitrate tetrahydrate (Ca(NO3)2·4H2O, and ammonium phosphate dibasic ((NH4)2HPO4) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. Moreover, α-MEM (Minimum Essential Medium Eagle-α), 10% fetal bovine serum (FBS), and antibiotics (penicillin/streptomycin) used for the cell experiment were purchased from Gibco BRL (Invitrogen Co., Carlsbad, CA, USA). Cell proliferation and activity were evaluated, using CCKi-8 (Enzo Life Science Inc., New York, NY, USA) and a TRACP & ALP assay kit (TakaRa, Kyoto City, Kyoto Prefecture, Japan).
7
Analysis of Osteoclast Differentiation
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After induction of osteoclast differentiation, the cells were fixed with 4% PFA for 10 min and washed three times with PBS. The differentiated osteoclasts were analyzed using TRAP staining and activity assay with an Acid-Phosphatase Kit (Sigma-Aldrich; St. Louis, MO, USA; 387A). TRAP-positive cells, including three or more multinucleated nuclei, were calculated under microscopes (KERN & SOHN GmbH and Korea Lab Tech, Balingen Germany). Osteoclastogenesis-induced monocytes were subjected to TRAP activity assay using TRACP & ALP Assay Kit (Takara Bio Inc.; Shiga, Japan) as described by the manufacturer’s protocol. The absorbance of TRAP activity was analyzed at 405 nm (BioTek, Seoul, Korea) and expressed as a percentage of the untreated control.
8
Runx2 Transfection and Osteogenic Differentiation
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For alkaline phosphatase (ALP) measurement, 0.3 μg of Runx2 mRNA or pDNA was transfected to MSCs cultured in 96-well plates, four times every 3 days. Fourteen days after the first transfection, ALP expression was measured using a TRACP & ALP assay kit (Takara Bio Inc., Shiga, Japan), which uses p-nitro-phenyl phosphate substrate to detect ALP enzymatic activity spectroscopically at an absorbance of 405 nm. For osteocalcin transcript measurement, 3 μg of Runx2 mRNA or pDNA was transfected to MSCs cultured in 12-well plates, four times every 3 days. Twenty-one days after the first transfection, total RNA in MSCs was extracted using an RNeasy mini kit, followed by quantitative real-time PCR using an ABI Prism 7500 sequence detector (Applied Biosystems, Foster City, CA, USA), and TaqMan gene expression assays (Applied Biosystems; Hs01587814_g1 for osteocalcin and Hs01060665_g1 for β-actin). In these osteogenic differentiation experiments, MSCs were cultured in MSC Go Rapid Osteogenic XF (Biological Industries, Beit Haemek, Israel), after the first transfection.
9
Osteogenic Differentiation of hMSCs
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hMSCs were used in in vitro experiments by considering the future clinical application using hMSCs. hMSCs were seeded onto micropatterned plates for spheroid culture and onto conventional plates for monolayer culture at 400,000 cells/well in 12-well plates and 40,000 cells/well in 96-well plates, respectively. At twenty-four hours after Runx2 and GFP transfection, the MSCs were cultured in osteogenic medium (50 μg/mL ascorbic acid phosphate [AsAP], 10 mM β-glycerophosphate [β-GP], and 0.1 μM dexamethasone [Dex], DMEM [high glucose]-containing 10% FBS, and 1% penicillin/streptomycin). At 14 days after incubation with osteogenic medium, the ALP activity of MSCs in the 96-well plates was assayed using the TRACP&ALP Assay Kit (Takara Bio) according to the manufacturer’s protocol. The kit uses p-NPP as a phosphatase substrate, which turns yellow when dephosphorylated by ALP. ALP activity was measured by the absorbance at 405 nm after color formation. At 28 days after incubation with osteogenic medium, total mRNA was extracted from the MSCs in the 12-well plates using RNeasy Mini Kits (QIAGEN), according to the manufacturer’s protocol. Then, osteogenic differentiation was evaluated by real-time qPCR for osteocalcin.
10
Murine Osteoclastogenesis Protocol
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Gold (III) chloride hydrate (99.999% trace metals basis), sodium citrate, red blood cell lysing buffer, and acid phosphatase, leukocyte acid phosphatase (TRAP) kits were purchased from Sigma-Aldrich (St Louls, MO). TRACP&ALP Assay kit was purchased from Takara (Seoul, Korea). Alendronate sodium trihydrate was purchased from TCI (Tokyo Chemical Industry Co., LTD, Japan). Macrophage colony stimulating factor (M-CSF) and recombinant murine sRANK ligand were purchased from Peprotech Korea (Seoul, Korea). RIPA lysis buffer and PBS 10X were purchased from Millipore. Protease inhibitor cocktail tablets were purchased from Roche Diagnostics Indianapolis (USA). ICR six-week old male mice, used for extraction of BMMs, were purchased from Young bio (Sung-nam, Korea). Minimum Essential Media (MEM) Alpha Medium, fetal bovine serum (FBS), antibiotic agents (penicillin/streptomycin, PS) and Dulbecco’s Phosphate Buffered Saline (DPBS) were purchased from GIBCO BRL (Invitrogen Co., USA). 48-well cell culture plates and cell culture dishes (100 mm × 20 mm) were purchased from Corning Incorporated (New York, USA). EZ-Cytox (enhanced cell viability assay kit) was purchased from Dogen (Seoul, Korea).
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