In order to examine extracellular ALP activity, an Alkaline Phosphatase Colorimetric Assay Kit (Abcam) was used in accordance with the manufacturer's protocol. In the assay, the p‐nitrophenyl phosphate was used as a phosphatase substrate. Obtained product was measured at 405 nm wavelength (Epoch; BioTek). The amount of pNP was obtained by sample readings applied to a standard curve. ALP activity was calculated using the following formula: ALP activity (U/mL) = A/V/T (where A—pNP amount; V—volume of sample added to well (mL); T—reaction time).
Alkaline phosphatase assay kit colorimetric
Manufactured by Abcam
Sourced in United Kingdom, United States
The Alkaline Phosphatase Assay Kit (Colorimetric) is a laboratory tool designed to quantify the activity of the enzyme alkaline phosphatase in various sample types, including cell lysates and biological fluids. The kit utilizes a colorimetric method to measure the conversion of a specific substrate by the enzyme, providing a reliable and convenient way to assess alkaline phosphatase levels.
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Lab products found in correlation
Spin x uf concentrators, by Merck Group (2 mentions)
Fugene 6 reagent, by Promega (2 mentions)
Synergy mx, by Agilent Technologies (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Infinite m200 pro, by Tecan (2 mentions)
Spectrometer, by BMG Labtech (1 mentions)
Versamax microplate reader, by Molecular Devices (1 mentions)
Nbt bcip, by Thermo Fisher Scientific (1 mentions)
Bz x700 series microscope, by Keyence (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Model synergy 2, by Agilent Technologies (1 mentions)
Synergy lx multi mode reader, by Agilent Technologies (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
α sma, by Cell Signaling Technology (1 mentions)
Miscript sybr green pcr kit, by Qiagen (1 mentions)
Real time pcr primers, by Sangon (1 mentions)
Imarktm microplate reader, by Bio-Rad (1 mentions)
Spectramax 190, by Molecular Devices (1 mentions)
Alp assay buffer, by Abcam (1 mentions)
Mithras, by Berthold Technologies (1 mentions)
38 protocols using alkaline phosphatase assay kit colorimetric
1
Extracellular ALP Activity Assay
2
Quantifying Extracellular ALP Activity
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Extracellular activity of ALP was determined in the supernatants collected after 7, 14, and 18 days of osteogenic culture on investigated surfaces. The assay was performed with an Alkaline Phosphatase Colorimetric Assay Kit (Abcam, Cambridge, UK), according to the protocol attached by the manufacturer. The test samples were prepared from each culture in duplicate and were diluted twofold. Sample background control was included and the background was corrected by subtracting the value derived from the zero standards from all standards, samples, and sample background control. In the reaction, the p-nitrophenyl phosphate (pNPP) was used as a phosphatase substrate. The substrate was hydrolyzed into para-nitrophenol (pNP) by alkaline phosphatase. The product of the reaction was measured at 405 nm wavelength using spectrometer (BMG Labtech, Ortenberg, Germany). Sample readings were applied to the standard curve to obtain the amount of pNP generated by the ALP sample (glycine units). The enzymatic activity was determined using the following formula.
ALP activity (μU/mL) = A/V/T, where (i) A is the amount of pNP generated by the samples (in μmol); (ii) V is the volume of sample added to the assay well (in mL); and (iii) T is the reaction time.
ALP activity (μU/mL) = A/V/T, where (i) A is the amount of pNP generated by the samples (in μmol); (ii) V is the volume of sample added to the assay well (in mL); and (iii) T is the reaction time.
3
Alkaline Phosphatase Colorimetric Assay
4
Quantifying Secreted Proteins and ALP Activity
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To evaluate the extracellular levels of secreted proteins, ELISA was performed. In order to evaluate the amount of Tumour protein p53 (p53), VEGF, IL‐1α and IL‐1β, supernatants were collected after 7th day from cells cultured in control medium. To evaluate the extracellular level of BMP‐2 and alkaline phosphatase (ALP), culture medium from 11th day of osteogenic differentiation was collected. All ELISA test were purchased from MyBioSource (San Diego, California, USA) and performed in accordance to manufacturer's instructions.
To estimate extracellular ALP activity, an Alkaline Phosphatase Colorimetric Assay Kit (Abcam) was used in according to manufacturer's protocol. Briefly, in the assay, the p‐nitrophenyl phosphate was used as a phosphatase substrate. The substrate was hydrolysed into p‐nitrophenol by ALP. The product was then measured spectroscopically at 405 nm wavelength (BMG Labtech). The amount of pNP was obtained by sample readings applied to a standard curve. ALP activity was calculated using the following formula: ALP activity (U/ml) = A/V/T (where A ‐ pNP amount; V ‐ volume of sample added to well (ml); T ‐ reaction time).
To estimate extracellular ALP activity, an Alkaline Phosphatase Colorimetric Assay Kit (Abcam) was used in according to manufacturer's protocol. Briefly, in the assay, the p‐nitrophenyl phosphate was used as a phosphatase substrate. The substrate was hydrolysed into p‐nitrophenol by ALP. The product was then measured spectroscopically at 405 nm wavelength (BMG Labtech). The amount of pNP was obtained by sample readings applied to a standard curve. ALP activity was calculated using the following formula: ALP activity (U/ml) = A/V/T (where A ‐ pNP amount; V ‐ volume of sample added to well (ml); T ‐ reaction time).
5
Recombinant Semaphorin 4D Production
6
Evaluating Osteogenic Differentiation of hMSCs
7
Measuring Osteoblast ALP Activity
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We followed the procedure described by Berbéri et al., 2016 [24 (link)]. Expression of ALP, a typical osteoblast marker, was measured using an Alkaline Phosphatase Colorimetric Assay Kit (Abcam plc, Cambridge, UK) which uses p-nitrophenyl phosphate (pNPP) as a phosphatase substrate. Intracellular activity of ALP was assessed in control and experimental cultures at 7, 14, and 21 days after treatment. Briefly, cells from both groups were washed with PBS and lysed using ALP assay buffer. Thereafter, a total of 80 μL of the cell lysate was added to 50 μL pNPP in a 96-well plate, and the samples were shielded from direct light for 1 h at room temperature. Following that, 20 μL stop solution (3N NaOH) was added to the wells and the plate was read at 405 nm using an Enzyme Linked Immunosorbent Acid (ELISA) plate reader. Results were expressed as nM p-NP/ml/min and normalized to protein content as measured by the Lowry method in corresponding wells. The Lowry method is used to estimate the amount of protein in a given sample; the total protein concentration is displayed by a color change of the sample solution in proportion to protein concentration.
8
Osteogenic Differentiation Assays for hMSCs
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To confirm osteogenic differentiation and to determine the level of activity of the differentiated hMSCs, two assays were performed: alkaline phosphatase (ALP) activity and total protein content (micro-BCA assay). Alkaline phosphatase activity was assessed using the Alkaline Phosphatase Colorimetric Assay Kit (Abcam ab83369). According to standard protocols, after the cell's exposure to osteogenic differentiation medium for periods of 3 days, cells were washed twice with PBS. Then, 50 μL of the cell lysate with assay buffer was added to a 96 well-plate and 50 μL p-nitrophenyl phosphate (pNPP). The samples were incubated at 25 °C for 60 min and protected from light. In the last step, 20 μL stop solution was added to the wells, then; the plate was read at 405 nm in a microplate reader (Synergy LX Multi-Mode Reader from BioTek® (Model SLXFA). Alkaline phosphatase (ALP) activity was normalized by total protein content (micro-BCA assay). The total protein content was determined according to the protocol of the manufacture 150 μL of sample was placed in a 96 well-plate with 150 μL of working reagent made from a micro-BCA protein assay kit (Thermo Scientific). The well plate was covered with foil and incubated at 37 °C for 2 h. Absorbance was read at 562 nm using a BioTek Multi-Mode Microplate Reader (Model Synergy™ 2).
9
Osteoblast Mineralization and ALP Assay
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Alizarin Red S staining was performed on mature osteoblast monolayers to analyze mineralization. Briefly, monolayers were fixed in 4% PFA and stained with 40 mM Alizarin Red S for 30 minutes. Staining intensity was calculated relative to Alizarin Red S standards at a wavelength of 405 nm. Alkaline phosphatase activity was determined on mature osteoblast protein lysates using the alkaline phosphatase colorimetric assay kit from Abcam. Analysis was performed on three samples per condition.
10
Production of Recombinant Semaphorin 4D
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Recombinant Semaphorin 4D was produced as a secreted protein fused with AP. To produce recombinant Semaphorin, human embryonic kidney HEK293T cells were transfected with Sema4D-AP in pcDNA3 vector47 (link), using Fugene 6 reagent according to the manufacturer’s protocol (Promega) The supernatant of transfected cells was collected, filtered and concentrated 10-fold by Spin-X UF concentrators with a 100kDa cutoff (Millipore). Protein concentration was normalized to a range of 3–6 nM by measurement of the AP-activity using Alkaline Phosphatase Colorimetric Assay kit (Abcam).
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