RAW 264.7 cells (1 × 104 cells/well) were cultured with or without RANKL (50 ng/mL) in the presence of ISE in 24-well plates. The medium was replaced every 2 days with fresh medium. The TRACP & ALP double-stain kit (Takara Bio Inc., Shiga, Japan) was used to identify osteoclasts, following the manufacturer’s instructions, following incubation of RAW 264.7 cells for 5 days. We considered TRAP positive multinucleated cells (MNCs) containing 3 or more nuclei as osteoclast-like cells and counted and captured them by a microscope. The cell culture medium was used to measure TRAP activity using a TRACP & ALP double-stain kit (Takara Bio Inc.) with a microplate reader at 405 nm.
Tracp alp double stain kit
Manufactured by Takara Bio
Sourced in Japan
The TRACP & ALP double-stain kit is a laboratory tool designed for the simultaneous detection and visualization of Tartrate-Resistant Acid Phosphatase (TRACP) and Alkaline Phosphatase (ALP) activities in cells. The kit provides the necessary reagents and protocols to perform this dual-staining procedure.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Mtt reagent, by Avantor (1 mentions)
Ab45039, by Abcam (1 mentions)
Microplate spectrometer, by Bio-Rad (1 mentions)
Osteoblast inducer reagent, by Takara Bio (1 mentions)
Rpmi medium, by Merck Group (1 mentions)
Recombinant myostatin, by R&D Systems (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Eclipse ti e, by Nikon (1 mentions)
Cetylpyridinium chloride monohydrate, by Merck Group (1 mentions)
Methyl green, by Takara Bio (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Silver nitrate, by Merck Group (1 mentions)
P nitrophenyl phosphate pnpp, by Merck Group (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Bca protein assay method, by Thermo Fisher Scientific (1 mentions)
Quikchange xl site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Alizarin red s solution, by Merck Group (1 mentions)
Magnafire 4, by Optronics (1 mentions)
Ix70 microscope, by Olympus (1 mentions)
17 protocols using tracp alp double stain kit
1
Osteoclast Differentiation Assay
2
Immunostaining of Osteocytic Markers in Cultured Cells
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Outgrown cells cultured for 3, 7, and 10 days were used to analyze protein production. Prior to immunostaining, the medium was removed from the culture wells. Cells were then washed two times using PBS, followed by fixation with 4% paraformaldehyde (PFA) for 10 min. After fixation, cells were incubated with 0.02 mg·mL−1 phycoerythrin (PE) anti-mouse podoplanin (E11) antibody (BioLegend, CA, USA) for 20 min and washed with PBS three times. For sclerostin and alkaline phosphatase (ALP) staining, the fixed cells were permeabilized using 0.1% triton x-100 for 10 min. To block unspecific antibody binding, cells were incubated in 3% BSA for 1 h at room temperature. After blocking cells were then incubated overnight at 4 °C with a goat anti-mouse sclerostin antibody (affinity purified polyclonal antibody, RnD systems, MN, USA) or ALP antibody (RnD systems) followed by secondary staining with a fluorescence-conjugated antibody (rhodamine AffiniPure rabbit anti-goat IgG, Jackson ImmunoResearch Laboratories, PA, USA). ALP expression was also tested by TRACP&ALP double-stain Kit (TaKaRa, Shiga, Japan). In this staining, ALP-positive cells presented with a light purple stain. Early osteocytic cell line (MLO-A5) used as ALP-positive/sclerostin-negative control. Normal goat IgG (RnD systems) was used as negative control.
3
Rosiglitazone Modulates Osteoclast Differentiation
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To observe osteoclast differentiation, RAW264.7 cells were cultured in 96‐well plates in medium containing various concentrations of rosiglitazone in the presence 50 μg·mL−1 of RANKL. After 5 days, cells were stained for TRAP with a commercial kit (TRACP & ALP double‐stain kit, Takara, Shiga, Japan). Cytotoxicity of rosiglitazone was assayed using an MTT reagent (Amresco, Inc., Solon, OH, USA). In this case, cells were treated for 1 or 5 days without RANKL addition.
4
Osteogenic Differentiation of Cells
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The cells were cultured on each film for 1 day before treatment with
osteogenic differentiation medium. After allowing 1 day for cell adhesion to
take place, osteogenic differentiation medium (10 mM β-glycerophosphate,
50 µg/ml ascorbic acid, and 100 nM dexamethasone in growth medium) were
added to the cell culture film for 21 days. The ALP staining was performed
with Takara TRACP&ALP double-stain kit following the manufacturer’s
protocol (Takara Bio, Kyoto, Japan).
osteogenic differentiation medium. After allowing 1 day for cell adhesion to
take place, osteogenic differentiation medium (10 mM β-glycerophosphate,
50 µg/ml ascorbic acid, and 100 nM dexamethasone in growth medium) were
added to the cell culture film for 21 days. The ALP staining was performed
with Takara TRACP&ALP double-stain kit following the manufacturer’s
protocol (Takara Bio, Kyoto, Japan).
5
Femur Histology and Immunohistochemistry
6
Osteoblast Differentiation and Mineralization
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MC3T3-E1 cells, an osteoblastic cell line, were grown in RPMI medium (Sigma) supplemented with 10% FBS, 100 IU/mL penicillin and 100 mg/mL streptomycin at 37 °C in 5% CO2 in humidified air. Osteoblast differentiation was induced by Osteoblast-Inducer Reagent (Takara Bio Inc., Shiga, Japan), a cocktail of L-ascorbic acid, dexamethasone and β-glycerophosphoric acid in the presence or absence of CGRP (10, 100 nM) for 10 days. The culture medium was replaced every 3 days. Alkaline phosphatase (ALP) activity was determined by the TRACP & ALP double-stain kit (Takara Bio Inc.) according to the manufacturer’s protocol. Results were normalized to total cellular protein contents. The protein concentration of the lysates was determined using Pierce™ BCA Protein Assay Kit (Thermo Scientific, Rockford, IF, USA). Mineralization was determined using Alizarin red staining. The cells were fixed with 4% PFA and stained with Alizarin red to detect calcification. For quantification, cells were lysed and the absorbance was read with a microplate spectrometer (Bio-Rad Laboratories).
7
Osteogenic Differentiation of MSCs
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To evaluate osteogenic differentiation, MSCs were seeded at a density of 5000 cells/cm2 in a 24-well cell culture plate and cultured until 90% confluence. After 21 days of osteogenic induction, ALP and Alizarin Red S staining were performed using a TRACP & ALP double-stain Kit (TAKARA BIO Inc., Shiga, Japan) and Calcification Evaluation Set (Iwai Chemicals Co., Ltd., Tokyo, Japan), according to the manufacturers’ instructions, respectively.
8
Myostatin Regulates Osteoblast ALP
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hFOB cells were incubated in the osteogenic medium, supplemented with various concentrations of recombinant myostatin (R&D Systems), which was a mature form of myostatin. ALP staining was performed via the TRACP & ALP double‐stain Kit (TaKaRa Bio, Shiga, Japan). For ALP staining, cells were rinsed twice with PBS and then fixed in fixation buffer. After washed by distilled water, cells were stained with ALP substrate. The experiments were performed in duplicate for each condition.
9
Osteoblastic Differentiation Dynamics
10
Osteogenic Differentiation Assays
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Cells were seeded into 24-well plates at a concentration of 5 × 104 cells/well. After 24 h, the media were replaced with 25% of the different extracted osteogenic mediums and cultured for 14 days. Alkaline phosphatase staining (ALP) was performed at day 10, and Von Kossa and alizarin red s staining were performed at day 14 [11 (link), 14 (link)]. For ALP staining, the cells were fixed and stained with TRACP & ALP double-stain kit (Takara Bio USA Inc., CA, USA). For alizarin red s staining, the cells were fixed with cold methanol and washed with deionized water. Alizarin red s solution (1% w/v) was incubated with the samples for 3 min, removed, and washed with DI water 3 times. The staining was solubilized with 10% cetylpyridinium chloride monohydrate (Sigma-Aldrich, MO, USA) solution and the absorbance was measured at 570 nm using a spectrophotometer. For Von Kossa staining, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, MO, USA) in PBS for 10 min. After rinsing with deionized water, 5% silver nitrate (Sigma-Aldrich, MO, USA) was added and the samples were exposed to UV light for 60 min. The cells were washed and rinsed with 5% sodium thiosulfate (JT Baker) 3 times before counterstaining with methyl-green (Takara Bio USA Inc., CA, USA).
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