ALP activity, a hallmark enzyme of mature osteoblasts, was examined using the ALP activity colorimetric assay kit (BioViSion, USA). First, the cells were washed twice with PBS and then extracted with RIPA buffer (Solarbio). The optical density at 405 nm was measured using Varioskan™ LUX multimode microplate reader (Thermo Fisher Scientific).
Alp activity colorimetric assay kit
Manufactured by Abcam
Sourced in United States
The ALP activity colorimetric assay kit is a laboratory tool used to quantify the activity of the enzyme alkaline phosphatase (ALP) in biological samples. The kit provides the necessary reagents and protocols to measure ALP activity colorimetrically.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (2 mentions)
Varioskan lux multimode microplate reader, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Alp reaction solution, by Takara Bio (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Bcip nbt alkaline phosphatase color development kit, by Beyotime (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
Pierce protein assay kit, by Thermo Fisher Scientific (1 mentions)
Nbt bcip staining kit, by CoWin Biotech (1 mentions)
Paraformaldehyde (pfa), by Sangon (1 mentions)
Model 680, by Bio-Rad (1 mentions)
10 protocols using alp activity colorimetric assay kit
1
Osteoblast ALP Activity Colorimetric Assay
2
Assaying Alkaline Phosphatase and Mineralization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ALP staining and activity were analyzed by using an ALP reaction solution (Takara Bio Inc., Tokyo, Japan) and ALP activity colorimetric assay kit (Biovision, Milpitas, CA, USA), respectively, as previously described [63 (link)]. ARS was analyzed by using a 2% ARS (Sigma-Aldrich) solution (pH 4.2) to monitor mineralization, as previously described [49 (link),63 (link)].
3
Quantifying Osteoblast ALP Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ALP activity was quantified using the ALP Activity Colorimetric Assay Kit (BioVision, Inc., Milpitas, CA, USA) with some modifications. The Cells were cultured in 24-well plates. On days 5 and 10 of osteoblastic differentiation, the wells were rinsed with PBS and fixed with a fixative solution (3.7% formaldehyde in 90% ethanol) for 30 s. The fixative was removed and replaced with a p-nitro-phenyl phosphate solution, and the cells were incubated in the dark for 1 h at room temperature. The optical density for each well was measured at 405 nm using a microplate reader (SpectraMax® M5, Molecular Devices, San Jose, CA, USA). ALP enzyme activity was normalized to the cell count.
4
Quantifying hMSC Osteogenic Differentiation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To quantify osteogenic differentiation of hMSCs, alkaline phosphatase (ALP) activity was assessed using an ALP activity colorimetric assay kit (BioVision, Milpitas, CA, USA). In the ALP assay, 50 μL of 5 mM p-nitrophenyl phosphate (pNPP) as a phosphatase substrate were added into each well containing 80 μL of the culture supernatant. The reaction was at room temperature for 1 h in the dark. To test both sample and background controls, the reaction was stopped by adding 20 μL of 0.2 N NaOH and OD was measured at 405 nm in a microplate reader. The osteogenic differentiation of hMSCs cultured on FHA-Mg scaffolds and culture dishes in the presence of osteogenic induction medium or complete culture medium was compared at days 0, 7, 14, and 21.
5
Alkaline Phosphatase Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ALP staining was performed using an ALP activity colorimetric assay kit (BioVision, Inc.) following the manufacturer's instructions. Briefly, cells were fixed with 70% ethanol at room temperature for 30 min and washed with PBST. Then, 50 µl 5 mM P-nitrophenyl phosphate (p-NPP) solution was added to test and control samples, and 10 µl ALP enzyme solution was added to each p-NPP Standard wells and incubated at room temperature for 15–30 min. Cells were gently washed three times with cold PBS. Subsequently, cells were lysed with 1% Triton X-100 (Sigma-Aldrich; Merck KGaA) and washed with deionized water. The absorbance of p-nitrophenol at a wavelength of 405 nm was measured using a microplate reader (Bio-Rad Laboratories, Inc.) according to the manufacturer's instructions.
6
Osteogenic Differentiation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Osteogenic differentiation was induced by supplementing with 100 nM dexamethasone, 200 μM l -ascorbic acid and 10 mM β-glycerophosphate (Sigma) into the growth medium. One week later, ALP staining was performed by BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, Shanghai, China) and ALP activity was analyzed by ALP activity colorimetric assay kit (BioVision, Milpitas, CA, USA). After incubation for two weeks, cells were dyed with 0.1% ARS (Sigma) and ARS quantification was examined as previously reported.20 (link) The images were saved at 100× magnification.
7
Osteogenic Differentiation Assessment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ALP staining was performed using a NBT/BCIP staining kit (CoWin Biotech, Beijing, China) as previously described [22 (link)]. After osteogenic induction for 7 days, cells were fixed in 4% paraformaldehyde for 10 min, and then ALP staining was performed following the manufacturer’s instructions.
A commercialized ALP activity colorimetric assay kit (BioVision, Milpitas, CA, USA) was used to analyze ALP activity. The cultured cells were washed with cold PBS, then lysed with 1% Triton X-100 (Sigma-Aldrich) and scraped into distilled water. The absorbance was detected at 405 nm. Total protein concentrations were determined by the bicinchoninic acid (BCA) method using the Pierce protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). ALP activity was calculated from the absorbance levels relative to the protein concentration.
A commercialized ALP activity colorimetric assay kit (BioVision, Milpitas, CA, USA) was used to analyze ALP activity. The cultured cells were washed with cold PBS, then lysed with 1% Triton X-100 (Sigma-Aldrich) and scraped into distilled water. The absorbance was detected at 405 nm. Total protein concentrations were determined by the bicinchoninic acid (BCA) method using the Pierce protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). ALP activity was calculated from the absorbance levels relative to the protein concentration.
8
Quantifying Alkaline Phosphatase Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Alkaline phosphatase (ALP) staining was performed following the protocol of the NBT/BCIP staining kit (Aibosi, Shanghai, China). The cultured cells were rinsed with PBS and fixed in 4% paraformaldehyde for 30 min. Then, the cell layer was washed three times with PBS and incubated in alkaline solution for 20 min at 37 °C. An ALP activity colorimetric assay kit (Biovision, Milpitas, CA, USA) was used to analyze ALP activity. The cultured cells were rinsed three times with PBS, followed by 1% Triton X-100, and then scraped into distilled water. After that, three cycles of freezing and thawing were done. Bicinchoninic acid (BCA) method was used to determine the total protein content in the same sample using the Pierce protein assay kit (Aibosi, Shanghai, China). ALP activity relative to the amount of total protein in the sample was calculated.
9
Alkaline Phosphatase Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After removing culture medium, cells were rinsed in pre-warmed PBS. For alkaline phosphatase (ALP) activity detection, ALP activity colorimetric assay kit was available from BioVision (USA). The rinsed cells were lysed in 1% Triton X-100, scraped into distilled water, and finally assayed by microplate reader at 405 nm. For ALP staining, BMSCs were fixed by 4% paraformaldehyde (Sangon Biotech, China) and then stained in line with the direction (Gefan Biotechnology, China). Images were acquired using a light microscope (Leica Microsystems).
10
Alkaline Phosphatase Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ALP activity in cells and cell sheets cultured in 100 × 20-mm cell culture dishes was measured using an ALP activity colorimetric assay kit (Biovision, San Francisco, CA, USA). Cells were washed with DPBS, detached using a cell scraper, and sonicated in 300 µl of assay buffer. After sonication, the solution was centrifuged at 13,000 × g for 3 min at 4°C, and 80 µl of the supernatant was added to 50 µl of p-nitrophenyl phosphate substrate in a 96-well plate followed by incubation for 1 h at room temperature (22-25°C) in the dark. Stop solution (20 µl) was added to the colored samples, and the absorbance of each well was measured at 415 nm with a microplate reader (model 680; Bio-Rad, Hercules, CA, USA).
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!