Title: Fluorescence-coupled micropipette aspiration assay to examine calcium mobilization caused by red blood cell mechanosensing

Abstract

Abstract 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 onlymore » 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.« less

Authors:

Wang, Haoqing; Obeidy, Peyman; Wang, Zihao; Zhao, Yunduo; Wang, Yao; Su, Qian Peter; Cox, Charles D.; Ju, Lining Arnold

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• Search DOE PAGES for ORCID "0000-0002-7591-0864"

Publication Date:

Mon Mar 14 00:00:00 EDT 2022

Sponsoring Org.:

USDOE

OSTI Identifier:

1854627

Grant/Contract Number:  

BBIP

Resource Type:

Published Article

Journal Name:

European Biophysics Journal

Additional Journal Information:

Journal Name: European Biophysics Journal Journal Volume: 51 Journal Issue: 2; Journal ID: ISSN 0175-7571

Publisher:

Springer Science + Business Media

Country of Publication:

Germany

Language:

English