Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

ALP staining and activity

PX Pengcheng Xiao
ZZ Zhenglin Zhu
CD Chengcheng Du
YZ Yongsheng Zeng
JL Junyi Liao
QC Qiang Cheng
HC Hong Chen
CZ Chen Zhao
WH Wei Huang
ask Ask a question
Favorite

For ALP staining, cells were fixed with 4% paraformaldehyde for 30 min. The cells were then washed twice with PBS and stained using the BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime). Staining was observed under a bright field microscope after 30 min [32, 38].

For the measurement of ALP activity, cells were washed twice with PBS and lysed with 150 μL of NP-40 lysis buffer (Beyotime). The cell lysates were quantified by an alkaline phosphatase assay kit (Beyotime) using p-nitrophenyl phosphate (pNPP) as the substrate. In the presence of magnesium ions, pNPP was hydrolyzed by phosphatases to phosphate and p-nitrophenol. The rate of p-nitrophenol liberation is proportional to ALP activity and can be measured photometrically. The ALP activity was measured by a microplate reader (Thermo Scientific™, USA) at an absorbance of 405 nm [39]. In addition, the protein concentration of the cell lysate was determined by a BCA Protein Assay Kit (Beyotime), and ALP activity was normalized to total protein per well.

Copyright and License information: The Author(s) ©2021
Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A