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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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.

  1. Activation of glass-bottom dishes or coverslips
    1. Prepare the b.......

Representative Results

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 cells17 and Ref52 rat embryonic fibroblasts (Figure 5) move towar.......

Discussion

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.......

Acknowledgements

None.

....

Materials

NameCompanyCatalog NumberComments
Acrylamide 40 % National DiagnosticEC-810
Ammonium Persulfate FisherBP179-25
BD20A High frequency generatorElectro Technic Products12011A115 V - Handheld Corona Wand
Bind Silane (y-methacryloxypropyltrimethoxysilane) (Sigma AldrichM6514
Bis-acrylamide 2% National DiagnosticEC-820
Borosilicate glass capillariesWorld Precision Instruments1B100-4
Branson 2510 Ultrasonic CleanerBransonic40 kHz frequency
Coarse ManipulatorNarshigeMC35A
DMEMCorning10-013-CV
DMEM without phenol redSigma AldrichD5030
Dual-Stage Glass Micropipette PullerNarshigePC-10
Epidermal Growth FactorPeprotechAF-100-15
EthanolPharmco-aaper111000200
Fetal Bovine Serum (Qualified One Shot)GibcoA31606-02
Fibronectin EMD MilliporeFC010
Fluospheres Carboxylate 0.2 um InvitrogenF8810, F8807, F8811
Fugene 6Roche18150911.5 ug DNA / 6uL fugene 6 per 35mm dish
Glacial Acetic AcidFisher ChemicalA38SI-212
Glass Bottom DishCellVisD60-60-1.5-N
Glass CoverslipElectron Microscopy Sciences72224-0122 mm, #1.5
HClJT Baker9535-03
Hellmanex III Special cleaning concentrateSigma AldrichZ805939Used at 2% in ddH2O for cleaning coverslips
HEPES powderSigma AldrichH3375Make 50mM HEPES buffer, pH 8.5
Intelli-Ray 400 Shuttered UV Flood LightUviton InternationalUV0338
IsopropanolFisher ChemicalA417-4
MicroforgeNarshigeMF900
MicromanipulatorNarshigeMHW3
Mineral OilSigma AldrichM5904
Nanopure Life Science UV/UF SystemBarnsteadD11931ddH2O
Nikon Eclipse TiNikon
OptiMEMInvitrogen31985062
Parafilm MBemis Company, IncPM-992
PBS139 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 AldrichP4056
Ref52Rat embryonic fibroblast cell line; Culture in DMEM + 10% FBS
Ringer's Buffer134 mM NaCl, 5.4 mM KCl, 1 mM MgSO4, 2.4 mM CaCl2, 20 mM HEPES, 5 mM D-Glucose, pH 7.4
SKOV-3American Type Culture CollectionCulture in DMEM + 10% FBS
Sulfo-SANPAH Covachem 12414-1
Tabletop Plasma CleanerHarrick PlasmaPDC-32G
TEMED Sigma AldrichT9281-50

References

  1. Acerbi, I., et al. Human breast cancer invasion and aggression correlates with ECM stiffening and immune cell infiltration. Integrative Biology (Camb. 7 (10), 1120-1134 (2015).
  2. Mekhdjian, A. H., et al.

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