fabrication of micropipettes for fluorescence aspiration assay to study live cell rbc mechanosensing

प्रतिदीप्ति माइक्रोपिपेट आकांक्षा का उपयोग करके एकल-कोशिका मेकेनोबायोलॉजी

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&#160;tension. These assays enable the measurement of various mechanical parameters, such as cell membrane&#160;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.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mechanical Parameter</td>
			<td>Description</td>
			<td>Seminal Approaches</td>
		</tr>
		<tr>
			<td>Cell Stiffness</td>
			<td>Measurement of a cell&#39;s mechanical rigidity and elasticity.</td>
			<td>Aspiration of the cell membrane and analysis of deformation response to the negative pressure20,21.</td>
		</tr>
		<tr>
			<td>Adhesion Strength</td>
			<td>Evaluation of how strongly cells adhere to surfaces.</td>
			<td>Application of controlled suction to detach adhered cells from a substrate2,22.</td>
		</tr>
		<tr>
			<td>Membrane Tension</td>
			<td>Assessment of the tension or stress within cell membranes.</td>
			<td>Measurement of the membrane deformation in response to applied pressure23,24.</td>
		</tr>
		<tr>
			<td>Viscoelastic Properties</td>
			<td>Characterization of a cell&#39;s combined viscous and elastic behavior.</td>
			<td>Analysis of the time-dependent deformation response to aspiration23,25.</td>
		</tr>
		<tr>
			<td>Deformability</td>
			<td>Determination of how easily a cell can change shape.</td>
			<td>Evaluation of the extent of deformation under controlled suction20,24.</td>
		</tr>
		<tr>
			<td>Surface Tension</td>
			<td>Measurement of the tension at the cell&#39;s surface.</td>
			<td>Assessment of the pressure required to form a micropipette membrane protrusion26.</td>
		</tr>
		<tr>
			<td>Cell-Material Interaction</td>
			<td>Study of interactions between cells and materials or substrates.</td>
			<td>Aspiration of cells in contact with different materials and observation of interactions2,24.</td>
		</tr>
		<tr>
			<td>Cell-Cell Interaction</td>
			<td>Examination of interactions between neighboring cells.</td>
			<td>Aspiration of a group of cells and analysis of their intercellular forces27.</td>
		</tr>
	</tbody>
</table>
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 &#181;m, 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.

लेखक स्पॉटलाइट: प्रतिदीप्ति microaspiration के माध्यम से लाइव-सेल Mechanobiology अग्रिम

प्रतिदीप्ति माइक्रोपिपेट आकांक्षा परख लाल रक्त कोशिका mechanosensing की जांच करने के लिए

The scope of our research is directed towards lifestyle mechanobiology, specifically the behavior of cells where mechanical stress is applied. We are investigating the intracellular signaling in single cells. The proposed FMPA system utilizes fluorescence imaging combined with mechanical stimuli.That is, the aspiration pressure. There are currently further advancements that have attempted to combine more than two techniques with the microaspiration setup like microfluidics or image analysis software. Current experimental challenges lie with the fact that the setup is quite labor intensive and is operator dependent.As the system is manually operated, there are inconsistencies that arise in specific steps. For example, filament preheating. The findings of research demonstrated there was a corresponding increase in the influx of calcium ions into the RBCs when the pressure was incrementally increased between negative 10 millimeter mercury to negative 40 millimeter mercury.This suggests that RBCs poses the ability to sense changes in their mechanical environment and respond by rapid calcium-related channel activities. 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.

लाइव सेल आरबीसी मेकेनोसेंसिंग का अध्ययन करने के लिए प्रतिदीप्ति आकांक्षा परख के लिए माइक्रोपिपेट का निर्माण

शुरू करने के लिए, एक P-1000 माइक्रोपिपेट पुलर पर एक बोरोसिलिकेट केशिका ट्यूब माउंट करें। 516 पर हीट सेट, 75 पर सेट वेग, 250 पर सेट समय और 500 पर दबाव सेट के साथ प्रीसेट प्रोग्राम का उपयोग करें। एक माइक्रोपिपेट कटर पर बंद समाप्त माइक्रोपिपेट में से एक को माउंट करें।एक बंद टिप खोलने के लिए, हीटिंग तापमान को लगभग 50 से 60 डिग्री सेल्सियस तक समायोजित करें। एक 10x ऐपिस के साथ, माइक्रोपिपेट का पता लगाएं। माइक्रोपिपेट को बोरोसिलिकेट ग्लास बीड के करीब ले जाने के लिए समायोजन नॉब्स का उपयोग करें।ऐपिस को 30x में बदलें, फिर माइक्रोपिपेट टिप को झुकाए बिना जितना संभव हो सके ग्लास बीड के करीब माइक्रोपिपेट को स्थानांतरित करें। गर्मी के साथ कांच मनका नरम करने के लिए हीटिंग पैडल पर कदम रखें। धीरे नरम मनका में कच्चे, बंद micropipette टिप डालें जब तक वांछित व्यास प्राप्त किया जाता है.कांच मनका शांत जाने के लिए पैर चप्पू रिलीज, तो धीरे बंद विंदुक पर एक स्पष्ट, सीधे कटौती के साथ एक माइक्रोमीटर व्यास micropipette निकालने.

Research

JoVE Journal

Biology

Fabrication of Micropipettes for Fluorescence Aspiration Assay to Study Live Cell RBC Mechanosensing

Fluorescence Micropipette Aspiration Assay to Investigate Red Blood Cell Mechanosensing

Micropipette aspiration assays have long been a cornerstone for the investigation of live-cell mechanics, offering insights into cellular responses to ...

Fluorescence Micropipette Aspiration Assay to Study Live Cell RBC Mechanosensing

प्रतिदीप्ति माइक्रोपिपेट आकांक्षा परख लाइव सेल आरबीसी मेकेनोसेंसिंग का अध्ययन करने के लिए