Naringin, β-glycerophosphate, L-ascorbic acid-2-phosphate, dimethyl Sulfoxide (DMSO), 1,25-dihydroxyvitamin D3, and Alizarin Red S were purchased from Sigma (St. Louis, MO, USA). Calcium Colorimetric Assay Kit and Alkaline Phosphatase Activity Colorimetric Assay Kit were obtained from Biovision, Inc. The anti-ERK antibody and anti-pERK antibody were purchased from Cell Signaling (Boston, MA, USA). U0126 was from Beyotime (Shanghai, China). All other chemicals were purchased from sigma (St, Louis, MO, eearch Ethical Committee of the USA).
Alkaline phosphatase activity colorimetric assay kit
Manufactured by Abcam
Sourced in United States
The Alkaline Phosphatase Activity Colorimetric Assay Kit is a laboratory tool used to measure the activity of the alkaline phosphatase enzyme. The assay kit utilizes a colorimetric method to quantify the enzyme's activity in samples.
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Lab products found in correlation
Multiskan go microplate spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Multiskan go, by Thermo Fisher Scientific (2 mentions)
Anti erk antibody, by Cell Signaling Technology (1 mentions)
Anti p erk antibody, by Cell Signaling Technology (1 mentions)
Calcium colorimetric assay kit, by Abcam (1 mentions)
L ascorbic acid 2 phosphate, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Alizarin red s, by Merck Group (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
Naringin, by Merck Group (1 mentions)
U0126, by Beyotime (1 mentions)
12 well plate, by BD (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Stempro osteogenesis differentiation kit, by Thermo Fisher Scientific (1 mentions)
Synergy 2 microplate reader, by Agilent Technologies (1 mentions)
Nanosight lm10, by Malvern Panalytical (1 mentions)
Flexstation 3 multi mode microplate reader, by Molecular Devices (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Phosphate saline buffer pbs, by Thermo Fisher Scientific (1 mentions)
26 protocols using alkaline phosphatase activity colorimetric assay kit
1
Osteoblast Differentiation Assay
2
Osteoblast Differentiation with PIN
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Osteoblast differentiation was induced using OS containing 50 μg/mL L-AA and 10 mM β-GP with PIN (10 and 30 μM) for 7 days. The cell lysates were performed according to the manufacturer’s protocol using alkaline phosphatase activity colorimetric assay kit (Biovision, Milpitas, CA, USA) [46 (link)].
3
Measuring Alkaline Phosphatase Activity
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Alkaline phosphatase (ALP) activity was measured after cells (1.5 × 104 cells/disc) were seeded onto control and Er,Cr:YSGG laser–treated disc surfaces in 12-well plates (Falcon) and incubated for 1, 3, and 5 days. We used highly sensitive, simple, direct and high-throughput screening ready colorimetric assay designed to measure ALP activity (Alkaline Phosphatase Activity Colorimetric Assay Kit, catalog no. 412-500; BioVision Inc., Milpitas, CA, USA). The absorbance of the samples was measured with a microplate reader at the optical density of 405 nm with a reference wavelength of 650 nm.
4
Alkaline Phosphatase Activity in Stem Cell Cultures
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Cell cultures were grown at 8×103 stem cells/mm in a 37°C humidified incubator containing 5% CO2 (Fig. 1 ) with α-MEM or osteogenic induction media (StemPro® Osteogenesis Differentiation kit; Gibco; Thermo Fisher Scientific, Inc.) were harvested on days 1, 3 and 7. Cells were detached using trypsin (Gibco; Thermo Fisher Scientific, Inc.) and washed twice with PBS for 1 min. Alkaline phosphatase activity assays were performed using the Alkaline Phosphatase Activity Colorimetric assay kit (cat no. K412-500; BioVision, Inc., Milpitas, CA, USA) according to the manufacturer's protocol. The cells were resuspended with assay buffer, sonicated at 70–80% intensity for 1 min at 4°C and then centrifuged at 13,000 × g for 3 min at 4°C to remove insoluble material. The supernatant was mixed with p-nitrophenylphosphate substrate (provided in the kit) and incubated at 25°C for 60 min. The optical density of the resultant p-nitrophenol at 405 nm was determined spectrophotometrically.
Stem-cell spheroids formed in the concave micromolds were moved to culture plates on day 3. The spheroids were grown with α-MEM or osteogenic induction media in a 37°C humidified incubator containing 5% CO2 (Fig. 1 ) and harvested on days 1, 3 and 7. Alkaline phosphatase activity assays were performed as described above on the spheroids.
Stem-cell spheroids formed in the concave micromolds were moved to culture plates on day 3. The spheroids were grown with α-MEM or osteogenic induction media in a 37°C humidified incubator containing 5% CO2 (
5
Osteogenic Differentiation of P-MSCs
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Osteogenesis was quantitated by measuring ALP activity. Passages 3–5 P-MSCs were grown in the osteogenic medium; the cultured cells were using 100 mM Tris and 1% Triton-100. Furthermore, the Alkaline Phosphatase Activity Colorimetric Assay Kit (BioVision) was used for determining the ALP activity at the second and third week of culture by using a previously described method [17 (link)]. This method employs the conversion of p-nitrophenyl phosphate to p-nitrophenol, determined at a wavelength of 450 nm, and the ALP activity is calculated from a standard value. In this study, the total p-nitrophenol formed was normalized to total protein in 1 hour to determine the ALP activity (ALP activity/min/mg protein). The fold change activity in the experimental and control groups at the second and third week of culture was also compared.
Because the ALP mRNA test revealed that P-MSCs significantly induced osteoblast differentiation at calcitriol concentrations of 10−8 M and 10−7 M, the ALP activity assay was attempted using these concentrations. The ALP activity of the control, Vit. C-p, 10−8 M calcitriol, and 10−7 M calcitriol groups was assessed at the second and third weeks of culture.
Because the ALP mRNA test revealed that P-MSCs significantly induced osteoblast differentiation at calcitriol concentrations of 10−8 M and 10−7 M, the ALP activity assay was attempted using these concentrations. The ALP activity of the control, Vit. C-p, 10−8 M calcitriol, and 10−7 M calcitriol groups was assessed at the second and third weeks of culture.
6
Quantifying Alkaline Phosphatase Activity
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In brief, 1 mL of 1% Triton X-100 solution was added to each hydrogel sample and the cells were lysed by performing three repeat freeze-thaw cycles. To measure intracellular ALP activity, 20 mL of cell lysate and 60 mL of ALP assay buffer were added to a 96-well plate. Subsequently, 50 mL of 5 mM p-nitrophenyl phosphate was added to each well containing hydrogel samples. ALP standards were prepared and added to the 96-well plate according to manufacturer protocol (Alkaline Phosphatase Activity Colorimetric Assay kit; BioVision, Waltham, MA, USA). The samples were incubated in the dark at 25 °C for 60 min to allow the reaction to take place. ALP activity was determined by measuring optical density at 405 nm using a Biotek Synergy 2 microplate reader (Santa Clara, CA, USA).
7
Quantifying Cellular and Extracellular Vesicle TNAP Activity
8
Alkaline Phosphatase Activity Assay
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The cell lysates were performed according to the manufacturer’s protocol using alkaline phosphatase activity colorimetric assay kit (Biovision, Milpitas, CA, USA) as previously described [50 (link)]. The absorbance was measured at 405 nm using the Multiskan GO microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
9
Assessing Smooth Muscle Cell ALP Activity
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SMCs were incubated for 7 days in the following conditions: DMEM alone. Forty-eight hours after incubation under the different media conditions, ALP activity in human aortic smooth muscle cell media was assessed using Alkaline Phosphatase Activity Colorimetric Assay Kit (BioVision) according to manufacturer protocols. Eighty microliters of media was added to each well. Eighty microliters of fresh media were also added to separate wells to use as a sample background control. Twenty microliters of Stop Solution was added to the background control. Then, 50 μL of 5 mM p-nitrophenyl phosphate (pNPP) was added to each well and reaction was incubated for 1 h at 25°C, protected from light. Twenty microliters of Stop Solution was added to each sample and standard well to stop the reactions. O.D. was measured at 405 nm in micro plate reader. Using standard curve, ALP activity in the media of each sample was calculated and is expressed in Glycine Units. Glycine Units are defined as the amount of enzyme causing hydrolysis of 1 μmol of pNPP per minute at pH 9.6 and 25°C.
10
Alkaline Phosphatase Activity Quantification
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The alkaline phosphatase activity (ALP) was determined after 7 days and 14 days of culture on cell lysates using a commercial kit (Alkaline Phosphatase Activity Colorimetric Assay Kit, BioVision; Milpitas, CA, USA), as presented in a previous paper [67 (link)]. Alkaline phosphatase activity was calculated according to the formula:
where A represents the amount of p-nitrophenol (pNP) expressed by the samples (in μmol), V is the volume of lysate used in the reaction (in mL), and T represents the reaction time (in minutes).
To avoid variations due to different protein concentrations, all samples were reported to the protein concentration which was previously determined using the Bradford method. Thus, the concentrations of the reaction product (p-nitrophenol) were normalized to 1 μg protein.
where A represents the amount of p-nitrophenol (pNP) expressed by the samples (in μmol), V is the volume of lysate used in the reaction (in mL), and T represents the reaction time (in minutes).
To avoid variations due to different protein concentrations, all samples were reported to the protein concentration which was previously determined using the Bradford method. Thus, the concentrations of the reaction product (p-nitrophenol) were normalized to 1 μg protein.
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