1. Pulling Glass Micropipettes
Equipment: Micropipette Puller, Microforge.
Glass: Boroscillicate glass capillaries (~1.5 mm external diameter, ~1.4 mm internal diameter).
<ol>
	<li>Micropipettes are pulled using the same basic approach that is used to prepare glass microelectrodes for electrophysiology recordings. Briefly, a glass capillary is heated in the middle and when the glass starts to melt the two halv...

<ol>
	<li>Rand, R. P., Burton, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+the+red+cell+membrane.+I.+Membrane+stiffness+and+intracellular+pressure.">Mechanical properties of the red cell membrane. I. Membrane stiffness and intracellular pressure.</a> Biophys. J. 4, 115-135 (1964).</li><li>Discher, D. E., Mohandas, N., Evans, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+maps+of+red+cell+deformation:+hidden+elasticity+and+in+situ+connectivity.">Molecular maps of red cell deformation: hidden elasticity and in situ connectivity.</a> Science. 266, 1032-1035 (1994).</li><li>Schmid-Sch&#246;nbein, G. W., Sung, K. 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Microaspiration provides a simple and highly reproducible method to estimate cell stiffness/deformability by applying negative pressure to a cell membrane and measuring membrane deformability in response to well-defined pressure. It was first developed by Mitchison and Swann (1954) to characterize the elastic properties of sea-urchin eggs to provide insights into the mechanisms of cell division 21 and then to look at the mechanical properties in red blood cells 1. This method has been used in multip...

<table border="1">
 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalog/Model Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Sutter pipette puller</td>
 <td>Sutter Instruments</td>
 <td>P-97</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microforge</td>
 <td>Narishige</td>
 <td>MF-830</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Inverted Fluorescent Microscope</td>
 <td>Zeiss</td>
 <td>Axiovert 200M</td>
 <td>The microscope should be preferably equipped with 3D/deconvolution capabilities.</td>
 </tr>
 <tr>
 <td>Videocamera</td>
 <td>Zeiss</td>
 <td>AxioCam MRm</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Image Acquisition sotware</td>
 <td>Zeiss</td>
 <td>AxioVision</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pneumatic Pressure Transducer</td>
 <td>BioTek</td>
 <td>DPM-1B</td>
 <td>DPM1B Pneumatic Transducer Tester can now be found by FLUKE.</td>
 </tr>
 <tr>
 <td>Pipette glass</td>
 <td>Richland</td>
 <td>Customized glass</td>
 <td>Pipettes were customized with a 1.2 inner diameter and 1.6 outer diameter.</td>
 </tr>
 <tr>
 <td>DiI Dye</td>
 <td>Invitrogen</td>
 <td>D282</td>
 <td>Dissolves well in DMSO</td>
 </tr>
 </tbody>
</table>

micropipette aspiration of substrate-attached cells to estimate cell stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Watch this Scientific Journal Video about Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness at JoVE.com

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

micropipette-aspiration-substrate-attached-cells-to-estimate-cell

Research

JoVE Journal

Bioengineering

19.7K vistas.  University of Illinois. Aquí se describe un método sencillo y rápido para medir la rigidez de la célula. El principio general de este enfoque consiste en medir la deformación de la membrana en respuesta a la bien definida la presión negativa aplicada a través de una micropipeta a la superficie celular. Este método proporciona una poderosa herramienta para estudiar las propiedades biomecánicas de los adjuntos sustrato células.