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 processes^1,2.

Over the past decade, micropipette-based aspiration assays have stood out as a versatile tool in studying diverse cellular....

Protocol

This protocol follows the guidelines of and has been approved by the Human Research Ethics Committee of the University of Sydney. Informed consent was obtained from the donors for this study.

1. Human RBC isolation

NOTE: Step 1.1 should be performed by a trained phlebotomist using a protocol that has been approved by the Institutional Review Board.

1. Withdraw 5 mL of blood from the median cubital vein using a 19 G butterfly needle.
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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.......

Materials

Name	Company	Catalog Number	Comments
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