Fluorescence Micropipette Aspiration Assay to Investigate Red Blood Cell Mechanosensing
Summary
The exploration of cellular behavior under mechanical stress is pivotal for advances in cellular mechanics and mechanobiology. We introduce the Fluorescence Micropipette Aspiration (fMPA) technique, a novel method combining controlled mechanical stimulation with comprehensive analysis of intracellular signaling in single cells. This technique investigates new in-depth studies of live-cell mechanobiology.
Abstract
Micropipette aspiration assays have long been a cornerstone for the investigation of live-cell mechanics, offering insights into cellular responses to mechanical stress. This paper details an innovative adaptation of the fluorescence-coupled micropipette aspiration (fMPA) assay. The fMPA assay introduces the capability to administer precise mechanical forces while concurrently monitoring the live-cell mechanotransduction processes mediated by ion channels. The sophisticated setup incorporates a precision-engineered borosilicate glass micropipette connected to a finely regulated water reservoir and pneumatic aspiration system, facilitating controlled pressure application with increments as refined as ± 1 mmHg. A significant enhancement is the integration of epi-fluorescence imaging, allowing for the simultaneous observation and quantification of cell morphological changes and intracellular calcium fluxes during aspiration. The fMPA assay, through its synergistic combination of epi-fluorescence imaging with micropipette aspiration, sets a new standard for the study of cell mechanosensing within mechanically challenging environments. This multifaceted approach is adaptable to various experimental setups, providing critical insights into the single-cell mechanosensing mechanisms.
Introduction
The unfolding discoveries in the world of cellular behaviors have accentuated the role of mechanical stimuli, such as tension, fluid shear stress, compression, and substrate stiffness, in dictating dynamic cellular activities such as adhesion, migration, and differentiation. These mechanobiological aspects are of paramount importance in elucidating how cells interact with and respond to their physiological environments, impacting various biological processes1,2.
Over the past decade, micropipette-based aspiration assays have stood out as a versatile tool in studying diverse cellular responses to mechanical stimuli. This technique offers valuable insights into the intrinsic mechanical properties of living cells at the single-cell level, including cellular elastic modulus, stiffness, and cortical tension. These assays enable the measurement of various mechanical parameters, such as cell membrane tension, pressure exerted on the cell membrane, and cortical tension (summarized in Table 1). Studying the aspirational forces has enriched our understanding of how they influence cellular functions and processes, particularly in the realm of membrane dynamics, including fragmentation, elongation, and budding3,4.
| Mechanical Parameter | Description | Seminal Approaches |
| Cell Stiffness | Measurement of a cell's mechanical rigidity and elasticity. | Aspiration of the cell membrane and analysis of deformation response to the negative pressure20,21. |
| Adhesion Strength | Evaluation of how strongly cells adhere to surfaces. | Application of controlled suction to detach adhered cells from a substrate2,22. |
| Membrane Tension | Assessment of the tension or stress within cell membranes. | Measurement of the membrane deformation in response to applied pressure23,24. |
| Viscoelastic Properties | Characterization of a cell's combined viscous and elastic behavior. | Analysis of the time-dependent deformation response to aspiration23,25. |
| Deformability | Determination of how easily a cell can change shape. | Evaluation of the extent of deformation under controlled suction20,24. |
| Surface Tension | Measurement of the tension at the cell's surface. | Assessment of the pressure required to form a micropipette membrane protrusion26. |
| Cell-Material Interaction | Study of interactions between cells and materials or substrates. | Aspiration of cells in contact with different materials and observation of interactions2,24. |
| Cell-Cell Interaction | Examination of interactions between neighboring cells. | Aspiration of a group of cells and analysis of their intercellular forces27. |
Table 1: Mechanical parameters characterized by the micropipette aspiration assay.
The micropipette-based aspiration technique has been widely used to study red blood cells (RBCs), assessing the deformability and various mechanical characteristics of RBCs, which is essential in understanding their function in the circulatory system. RBCs exhibit remarkable adaptability, preserving their mechanical versatility against deformation when navigating through the intricate capillary network and inter-endothelial clefts5,6. During this journey, RBCs must traverse through passages as narrow as 0.5-1.0 µm, subjecting themselves to a multitude of mechanical forces, including tension and compression7,8,9. They also have high sensitivity to the shear stress generated by blood flow during circulation10. These processes promote the activation of regulatory mechanisms involving calcium influx, a crucial signaling event with well-established roles in cellular responses to mechanical stimuli11,12. The complex mechanisms governing the calcium-mediated mechanosensing remain compelling subjects of ongoing investigation.
In this context, the fMPA stands as an effective approach to reveal the extent of calcium mobilization under precisely controlled mechanical forces, allowing for the simultaneous application of mechanical modulation (using the micropipette aspiration system) and visualization of calcium intensity (using fluorescent indicators). It particularly mimics the physiological scenario when the RBC travels through narrowing blood vessels. It is worth noting that the fMPA system we developed can generate pressure with a resolution of 1 mmHg. The implemented high-speed camera can achieve a temporal resolution of 100 ms and a spatial resolution at the submicron-meter level. These configurations ensure the precise application of mechanical forces to live cells and simultaneously capture the resulting cellular signaling. Moreover, due to the integrative engineered nature of this setup, the micropipette aspiration assay can be readily adapted to complement other equipment or techniques, enabling further exploration of the intricacies of cell mechanics. This versatility stands as an additional advantage of this approach.
Protocol
Representative Results
Discussion
Micropipette aspiration assays embody a refined methodology, deploying substantial pressure modulation, exact spatial orchestration, and reliable temporal discernment to probe the profound intricacies of cellular biomechanics. This study places particular emphasis on the application of fMPA as a crucial tool for unveiling the nuanced mechanosensitive responses showcased by RBCs under varying stimuli. The concurrent use of brightfield and fluorescence signals enabled a multifaceted exploration of cellular phenomena, advan…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We thank Nurul Aisha Zainal Abidin and Laura Moldovan for additional donor recruitment, blood collection, and phlebotomy support. We thank Tomas Anderson and Arian Nasser for organizing the equipment and reagents. This research was funded by the Australian Research Council (ARC) Discovery Project (DP200101970-L.A.J.); the National Health and Medical Research Council (NHMRC) of Australia Ideas Grant (APP2003904-L.A.J.); NHMRC Equipment Grant-L.A.J.; NSW Cardiovascular Capacity Building Program (Early-Mid Career Researcher Grant-L.A.J.); NSW CVRN-VCCRI Research Innovation Grant; Office of Global and Research Engagement (Sydney-Glasgow Partnership Collaboration Award-L.A.J.); L.A.J. is a National Heart Foundation Future Leader Fellow Level 2 (105863), and a Snow Medical Research Foundation Fellow (2022SF176).
Materials
| µManager | Micro-Manager | Version 2.0.0 | |
| 1 mL Syringe | Terumo | 210320D | Cooperate with the Microfil |
| 200 µL Pipette | Eppendorf | 3123000055 | Red clood cell preparation |
| 22 x 40 mm Cover Slips | Knittel Glass | MS0014 | Cell chamber assembly |
| 50 mL Syringe | Terumo | 220617E | Connect to the water tower |
| Calcium Chloride (CaCl2) | Sigma-Aldrich | C1016 | Tryode's buffer preparation – 12 mM NaHCO3, 10 mM HEPES, 0.137 M NaCl, 2.7 mM KCl, and 5.5 mM D-glucose supplemented with 1 mM CaCl2. Final pH = 7.2 |
| Centrifuge 5425 | Eppendorf | 5405000280 | Red clood cell preparation |
| Clexane | Sigma-Aldrich | 1235820 | To prevent clotting of the collected blood. 10,000 U/mL |
| DAQami | Diligent | ||
| Fluorescence light source | CoolLED | pE-300 | Micropipette aspiration hardware system |
| Glass capillary | Narishige | G-1 | Micropipette manufacture |
| Glucose | Sigma-Aldrich | G8270 | Tryode's buffer preparation – 12 mM NaHCO3, 10 mM HEPES, 0.137 M NaCl, 2.7 mM KCl, and 5.5 mM D-glucose supplemented with 1 mM CaCl2. Final pH = 7.2 |
| Hepes | Thermo Fisher | 15630080 | Tryode's buffer preparation – 12 mM NaHCO3, 10 mM HEPES, 0.137 M NaCl, 2.7 mM KCl, and 5.5 mM D-glucose supplemented with 1 mM CaCl2. Final pH = 7.2 |
| High speed GigE camera | Manta | G-040B | Micropipette aspiration hardware system |
| High speed pressure clamp | Scientific Instrument | HSPC-2-SB | Cooperate with the pressure pump |
| High speed pressure clamp head stage | Scientific Instrument | HSPC-2-SB | Cooperate with the pressure pump |
| Imaris | Oxford Instruments | ||
| Inverted Microscopy | Olympus | Olympus IX83 | Micropipette aspiration hardware system |
| Microfil | World Precision Instruments | MF34G-5 | 34 G (67 mm Long) Revome air bubble in the cut micropipette and test the opening of the pipette tip |
| Micropipette Puller | Sutter instrument | P1000 | Micropipette manufacture |
| Milli Q EQ 7000 Ultrapure Water Purification System | Merck Millipore | ZEQ7000T0C | Carbonate/bicarbonate buffer & Tryode's buffer preparation |
| Pipette microforge | Narishige | MF-900 | Micropipette manufacture |
| Potassium Chloride (KCl) | Sigma-Aldrich | P9541 | Tryode's buffer preparation – 12 mM NaHCO3, 10 mM HEPES, 0.137 M NaCl, 2.7 mM KCl, and 5.5 mM D-glucose supplemented with 1 mM CaCl2. Final pH = 7.2 |
| Pressue Pump | Scientific Instrument | PV-PUMP | Induce controlled pressure during experiment |
| Prime 95B Camera | Photometrics | Prime 95B sCMOS | Flourscent imaging |
| Rotary wheel remote unit | Sensapex | uM-RM3 | Control panel for micropipette position adjustment |
| Scepter 3.0 Handheld Cell Counter | Merck Millipore | PHCC340KIT | Automatic cell counter |
| Sodium Bicarbonate (NaHCO3) | Sigma-Aldrich | S5761 | Carbonate/bicarbonate buffer preparation – 2.65 g of NaHCO3 with 2.1 g of Na2CO3 in 250 mL of Mili Q water – Final pH = 8-9. |
| Sodium Carbonate (Na2CO3) | Sigma-Aldrich | S2127 | Carbonate/bicarbonate buffer preparation – 2.65 g of NaHCO3 with 2.1 g of Na2CO3 in 250 mL of Mili Q water – Final pH = 8-9. |
| Sodium Chloride (NaCl) | Sigma-Aldrich | S7653 | Tryode's buffer preparation – 12 mM NaHCO3, 10 mM HEPES, 0.137 M NaCl, 2.7 mM KCl, and 5.5 mM D-glucose supplemented with 1 mM CaCl2. Final pH = 7.2 |
| Sodium Phosphate Monobasic Monohydrate (NaH2PO4 • H2O) |
Sigma-Aldrich | S9638 | Tryode's buffer preparation – 12 mM NaHCO3, 10 mM HEPES, 0.137 M NaCl, 2.7 mM KCl, and 5.5 mM D-glucose supplemented with 1 mM CaCl2. Final pH = 7.2 |
| Touch screen control unit | Sensapex | uM-TSC | Control panel for micropipette position adjustment |
| X dry Objective | Olympus | Olympus 60x/0.70 LUCPlanFL | Micropipette aspiration hardware system |
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