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Mechanical forces are important for controlling cell migration. This protocol demonstrates the use of elastic hydrogels that can be deformed using a glass micropipette and a micromanipulator to stimulate cells with a local stiffness gradient to elicit changes in cell structure and migration.
Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate underlying a cell must be manipulated. While hydrogels with graded stiffness and long-term migration assays have proven useful in durotaxis studies, immediate, acute responses to local changes in substrate tension allow focused study of individual cell movements and subcellular signaling events. To repeatably test the ability of cells to sense and respond to the underlying substrate stiffness, a modified method for application of acute gradients of increased tension to individual cells cultured on deformable hydrogels is used which allows for real time manipulation of the strength and direction of stiffness gradients imparted upon cells in question. Additionally, by fine tuning the details and parameters of the assay, such as the shape and dimensions of the micropipette or the relative position, placement, and direction of the applied gradient, the assay can be optimized for the study of any mechanically sensitive cell type and system. These parameters can be altered to reliably change the applied stimulus and expand the functionality and versatility of the assay. This method allows examination of both long term durotactic movement as well as more immediate changes in cellular signaling and morphological dynamics in response to changing stiffness.
Over the past few decades, the importance of the mechanical properties of a cell’s environment has garnered increasing recognition in cell biology. Different tissues and extracellular matrices have different relative stiffnesses and, as cells migrate throughout the body, they navigate these changes, using these mechanical properties to guide them1,2,3,4,5,6,7. Cells use the stiffness of a given tissue to inform their motile behavior....
1. Fabrication of Deformable Polyacrylamide Hydrogels with Embedded Fluorescent Microspheres
NOTE: Directions describe polymerization of a 25 kPa hydrogel that is 22 μm in diameter and approximately 66 μm thick. Each or all of these parameters can be modified and directions to do so can be found in Table 1 and in the notes17.
By preparing micropipettes (Figure 1) and normalizing the force generation of the pulls (Figure 2 and Figure 3) as described above, optimal durotactic conditions have been identified for multiple cell lines. Using this technique, as outlined in Figure 4, both SKOV-3 ovarian cancer cells17Â and Ref52 rat embryonic fibroblasts (Figure 5) move towar.......
Demonstrated here is a repeatable, single-cell durotaxis assay that allows assessment of a cell’s ability to alter its migration behavior in response to acute mechanical cues. This technique can also be used in combination with fluorescence microscopy and appropriate fusion proteins or biosensors to examine subcellular signaling and cytoskeletal events within seconds of mechanical stimulation or over a longer timescale during durotactic movement. Understanding a cell’s relationship to its environment involves.......
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....Name | Company | Catalog Number | Comments |
Acrylamide 40 %Â | National Diagnostic | EC-810 | |
Ammonium Persulfate | Fisher | BP179-25 | |
BD20A High frequency generator | Electro Technic Products | 12011A | 115 V -Â Handheld Corona Wand |
Bind Silane (y-methacryloxypropyltrimethoxysilane) ( | Sigma Aldrich | M6514 | |
Bis-acrylamide 2%Â | National Diagnostic | EC-820 | |
Borosilicate glass capillaries | World Precision Instruments | 1B100-4 | |
Branson 2510 Ultrasonic Cleaner | Bransonic | 40 kHz frequency | |
Coarse Manipulator | Narshige | MC35A | |
DMEM | Corning | 10-013-CV | |
DMEM without phenol red | Sigma Aldrich | D5030 | |
Dual-Stage Glass Micropipette Puller | Narshige | PC-10 | |
Epidermal Growth Factor | Peprotech | AF-100-15 | |
Ethanol | Pharmco-aaper | 111000200 | |
Fetal Bovine Serum (Qualified One Shot) | Gibco | A31606-02 | |
Fibronectin | EMD Millipore | FC010 | |
Fluospheres Carboxylate 0.2 um | Invitrogen | F8810, F8807, F8811 | |
Fugene 6 | Roche | 1815091 | 1.5 ug DNA / 6uL fugene 6 per 35mm dish |
Glacial Acetic Acid | Fisher Chemical | A38SI-212 | |
Glass Bottom Dish | CellVis | D60-60-1.5-N | |
Glass Coverslip | Electron Microscopy Sciences | 72224-01 | 22 mm, #1.5 |
HCl | JT Baker | 9535-03 | |
Hellmanex IIIÂ Special cleaning concentrate | Sigma Aldrich | Z805939 | Used at 2% in ddH2O for cleaning coverslips |
HEPES powder | Sigma Aldrich | H3375 | Make 50mM HEPES buffer, pH 8.5 |
Intelli-Ray 400 Shuttered UV Flood Light | Uviton International | UV0338 | |
Isopropanol | Fisher Chemical | A417-4 | |
Microforge | Narshige | MF900 | |
Micromanipulator | Narshige | MHW3 | |
Mineral Oil | Sigma Aldrich | M5904 | |
Nanopure Life Science UV/UF System | Barnstead | D11931 | ddH2O |
Nikon Eclipse Ti | Nikon | ||
OptiMEM | Invitrogen | 31985062 | |
Parafilm M | Bemis Company, Inc | PM-992 | |
PBS | 139 mM NaCl, 2.5 mM KCl, 28.6 mM Na2HPO4, 1.6 mM KH2PO4, pH 7.4 | ||
Platelet Derived Growth Factor-BB (PDGF-BB) | Sigma Aldrich | P4056 | |
Ref52 | Rat embryonic fibroblast cell line; Culture in DMEM + 10% FBS | ||
Ringer's Buffer | 134 mM NaCl, 5.4 mM KCl, 1 mM MgSO4, 2.4 mM CaCl2, 20 mM HEPES, 5 mM D-Glucose, pH 7.4 | ||
SKOV-3 | American Type Culture Collection | Culture in DMEM + 10% FBS | |
Sulfo-SANPAHÂ | Covachem | Â 12414-1 | |
Tabletop Plasma Cleaner | Harrick Plasma | PDC-32G | |
TEMEDÂ | Sigma Aldrich | T9281-50 |
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