Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

In vitro CD47 binding and blocking analyses

XG Xinjie Gao
HY Heng Yang
WX Weiping Xiao
JS Jiabin Su
YZ Yuwen Zhang
HW He Wang
WN Wei Ni
YG Yuxiang Gu
ask Ask a question
Favorite

As reported previously [24], 1 × 106 erythrocytes were incubated with biotinylated SIRPα WT/variants or anti-CD47 antibody for 30 min at 4 °C for cell binding analysis. The binding of biotinylated proteins was detected by Alexa Fluor 594-conjugated streptavidin as a secondary staining reagent. In cell blocking analysis, biotinylated SIRPα WT was incubated with Alexa Fluor 594-conjugated streptavidin to form SIRPα WT tetramers. Then, SIRPα WT tetramers were combined with CD47 antagonists added to CFSE+ erythrocytes simultaneously. After removal of the remaining nonspecific signals, the cells were analyzed by flow cytometry. Data for the above experiments represented the fluorescence intensity normalized to maximal binding and were fitted to a dose–response curve.

Copyright and License information: The Author(s) ©2022
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