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Fluorescence-coupled micropipette aspiration assay to examine calcium mobilization caused by red blood cell mechanosensing
Mechanical stimuli such as tension, compression, and shear stress play critical roles in the physiological functions of red blood cells (RBCs) and their homeostasis, ATP release, and rheological properties. Intracellular calcium (Ca(2+)) mobilization reflects RBC mechanosensing as they transverse th...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer International Publishing
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8964638/ https://www.ncbi.nlm.nih.gov/pubmed/35286429 http://dx.doi.org/10.1007/s00249-022-01595-z |
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author | Wang, Haoqing Obeidy, Peyman Wang, Zihao Zhao, Yunduo Wang, Yao Su, Qian Peter Cox, Charles D. Ju, Lining Arnold |
author_facet | Wang, Haoqing Obeidy, Peyman Wang, Zihao Zhao, Yunduo Wang, Yao Su, Qian Peter Cox, Charles D. Ju, Lining Arnold |
author_sort | Wang, Haoqing |
collection | PubMed |
description | Mechanical stimuli such as tension, compression, and shear stress play critical roles in the physiological functions of red blood cells (RBCs) and their homeostasis, ATP release, and rheological properties. Intracellular calcium (Ca(2+)) mobilization reflects RBC mechanosensing as they transverse the complex vasculature. Emerging studies have demonstrated the presence of mechanosensitive Ca(2+) permeable ion channels and their function has been implicated in the regulation of RBC volume and deformability. However, how these mechanoreceptors trigger Ca(2+) influx and subsequent cellular responses are still unclear. Here, we introduce a fluorescence-coupled micropipette aspiration assay to examine RBC mechanosensing at the single-cell level. To achieve a wide range of cell aspirations, we implemented and compared two negative pressure adjusting apparatuses: a homemade water manometer (− 2.94 to 0 mmH(2)O) and a pneumatic high-speed pressure clamp (− 25 to 0 mmHg). To visualize Ca(2+) influx, RBCs were pre-loaded with an intensiometric probe Cal-520 AM, then imaged under a confocal microscope with concurrent bright-field and fluorescent imaging at acquisition rates of 10 frames per second. Remarkably, we observed the related changes in intracellular Ca(2+) levels immediately after aspirating individual RBCs in a pressure-dependent manner. The RBC aspirated by the water manometer only displayed 1.1-fold increase in fluorescence intensity, whereas the RBC aspirated by the pneumatic clamp showed up to threefold increase. These results demonstrated the water manometer as a gentle tool for cell manipulation with minimal pre-activation, while the high-speed pneumatic clamp as a much stronger pressure actuator to examine cell mechanosensing directly. Together, this multimodal platform enables us to precisely control aspiration and membrane tension, and subsequently correlate this with intracellular calcium concentration dynamics in a robust and reproducible manner. |
format | Online Article Text |
id | pubmed-8964638 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Springer International Publishing |
record_format | MEDLINE/PubMed |
spelling | pubmed-89646382022-04-07 Fluorescence-coupled micropipette aspiration assay to examine calcium mobilization caused by red blood cell mechanosensing Wang, Haoqing Obeidy, Peyman Wang, Zihao Zhao, Yunduo Wang, Yao Su, Qian Peter Cox, Charles D. Ju, Lining Arnold Eur Biophys J Original Article Mechanical stimuli such as tension, compression, and shear stress play critical roles in the physiological functions of red blood cells (RBCs) and their homeostasis, ATP release, and rheological properties. Intracellular calcium (Ca(2+)) mobilization reflects RBC mechanosensing as they transverse the complex vasculature. Emerging studies have demonstrated the presence of mechanosensitive Ca(2+) permeable ion channels and their function has been implicated in the regulation of RBC volume and deformability. However, how these mechanoreceptors trigger Ca(2+) influx and subsequent cellular responses are still unclear. Here, we introduce a fluorescence-coupled micropipette aspiration assay to examine RBC mechanosensing at the single-cell level. To achieve a wide range of cell aspirations, we implemented and compared two negative pressure adjusting apparatuses: a homemade water manometer (− 2.94 to 0 mmH(2)O) and a pneumatic high-speed pressure clamp (− 25 to 0 mmHg). To visualize Ca(2+) influx, RBCs were pre-loaded with an intensiometric probe Cal-520 AM, then imaged under a confocal microscope with concurrent bright-field and fluorescent imaging at acquisition rates of 10 frames per second. Remarkably, we observed the related changes in intracellular Ca(2+) levels immediately after aspirating individual RBCs in a pressure-dependent manner. The RBC aspirated by the water manometer only displayed 1.1-fold increase in fluorescence intensity, whereas the RBC aspirated by the pneumatic clamp showed up to threefold increase. These results demonstrated the water manometer as a gentle tool for cell manipulation with minimal pre-activation, while the high-speed pneumatic clamp as a much stronger pressure actuator to examine cell mechanosensing directly. Together, this multimodal platform enables us to precisely control aspiration and membrane tension, and subsequently correlate this with intracellular calcium concentration dynamics in a robust and reproducible manner. Springer International Publishing 2022-03-14 2022 /pmc/articles/PMC8964638/ /pubmed/35286429 http://dx.doi.org/10.1007/s00249-022-01595-z Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/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/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Original Article Wang, Haoqing Obeidy, Peyman Wang, Zihao Zhao, Yunduo Wang, Yao Su, Qian Peter Cox, Charles D. Ju, Lining Arnold Fluorescence-coupled micropipette aspiration assay to examine calcium mobilization caused by red blood cell mechanosensing |
title | Fluorescence-coupled micropipette aspiration assay to examine calcium mobilization caused by red blood cell mechanosensing |
title_full | Fluorescence-coupled micropipette aspiration assay to examine calcium mobilization caused by red blood cell mechanosensing |
title_fullStr | Fluorescence-coupled micropipette aspiration assay to examine calcium mobilization caused by red blood cell mechanosensing |
title_full_unstemmed | Fluorescence-coupled micropipette aspiration assay to examine calcium mobilization caused by red blood cell mechanosensing |
title_short | Fluorescence-coupled micropipette aspiration assay to examine calcium mobilization caused by red blood cell mechanosensing |
title_sort | fluorescence-coupled micropipette aspiration assay to examine calcium mobilization caused by red blood cell mechanosensing |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8964638/ https://www.ncbi.nlm.nih.gov/pubmed/35286429 http://dx.doi.org/10.1007/s00249-022-01595-z |
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