सब्सट्रेट संलग्न कोशिकाओं के micropipette आकांक्षा सेल कठोरता का अनुमान लगाने के लिए
Summary
यहाँ हम सेल कठोरता को मापने के लिए एक त्वरित और आसान करने के लिए विधि का वर्णन है. इस दृष्टिकोण का सामान्य सिद्धांत के लिए अच्छी तरह से परिभाषित नकारात्मक दबाव कोशिका की सतह से एक micropipette के माध्यम से लागू करने के लिए जवाब में झिल्ली विरूपण उपाय है. इस विधि सब्सट्रेट संलग्न कोशिकाओं के biomechanical गुणों का अध्ययन करने के लिए एक शक्तिशाली उपकरण प्रदान करता है.
Abstract
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 μm 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.
Protocol
Discussion
यह Microaspiration एक सरल और अत्यधिक प्रतिलिपि प्रस्तुत करने योग्य एक कोशिका झिल्ली को नकारात्मक दबाव लागू करने और अच्छी तरह से परिभाषित दबाव के जवाब में झिल्ली विरूपता को मापने के द्वारा विधि सेल कठोरता / विरू?…
Disclosures
The authors have nothing to disclose.
Materials
| Name of reagent | Company | Catalog/Model Number | Comments |
| Sutter pipette puller | Sutter Instruments | P-97 | |
| Microforge | Narishige | MF-830 | |
| Inverted Fluorescent Microscope | Zeiss | Axiovert 200M | The microscope should be preferably equipped with 3D/deconvolution capabilities. |
| Videocamera | Zeiss | AxioCam MRm | |
| Image Acquisition sotware | Zeiss | AxioVision | |
| Pneumatic Pressure Transducer | BioTek | DPM-1B | DPM1B Pneumatic Transducer Tester can now be found by FLUKE. |
| Pipette glass | Richland | Customized glass | Pipettes were customized with a 1.2 inner diameter and 1.6 outer diameter. |
| DiI Dye | Invitrogen | D282 | Dissolves well in DMSO |
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