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Protocol

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Acknowledgements

Materials

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Bioengineering

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Published: April 4th, 2013

DOI:

10.3791/50310

1Centre for Vascular Research and Australian Centre for Nanomedicine, The University of New South Wales, 2School of Chemistry and Australian Centre for Nanomedicine, The University of New South Wales

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

Directed cell migration is a fundamental property of many cells and is a key aspect of many normal physiological processes, including embryonic development, defense against infection and wound healing. In addition, cell migration also plays a prominent role in many diseases such as vascular disease, tumour cell metastasis and chronic inflammation1,2. While the classical states of cell migration - polarization, protrusion extension, formation of adhesion, force generation and rear retraction - are generally accepted3,4 , the elucidation of spatiotemporal mechanisms by which signal integration is coordinated has been more challenging.

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1. Passivation of Glass Coverslips with PLL-PEG-biotin

This step is designed to passivate the surface so that cells adhere and migrate onto specific regions on the glass coverslip that are created with microcontract printing (Step 2-3a) or dip pen lithography (Step 3b). For the passivation, a co-polymer of poly-L-lysine (PLL) and polyethylene glycol (PEG) is used in which 20% of PEG molecules are grafted to biotin (PLL-PEG-biotin).

  1. To clean glass coverslips (18 mm x 18 mm), submerg.......

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To understand how cell integrate migratory signals25, we have developed a method to image cells with fluorescence microscopy that migrate in an environment with competing adhesive and soluble gradients (Figure 1). Adhesive tracks that contained fluorescent streptavidin and biotinylated RGD were created with microcontact printed tracks and dip pen lithography (Figure 2). Successful microcontact printing is indicated by the line profile of the fluorescence intensity across the t.......

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In this protocol, we used a commercially available microfluidic device (sticky-Slide Chemotaxis 3D from Ibidi) to study the effects of chemically modified surfaces and chemoattractant gradients on cell migration. This microfluidic setup does not require flow because the gradient is established by diffusion along the length of the channel. This is important because flow could differentially affect slowly migrating cells such as fibroblasts that form stable focal adhesions and fast migrating cells like most leuk.......

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The authors acknowledge funding from the Australian Research Council and National Health and Medical Research Council of Australia and also like to thank the Australian National Fabrication Facility for the SU-8 master for the microcontact printing. SHN is supported by the Ministry of Higher Education Malaysia and Universiti Sains Malaysia.

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Name Company Catalog Number Comments
Name of Reagent/Material Company Catalogue Number Comments
Coverglass staining outfits Thomas Scientific 8542 E40 Coverslip rack
Oven Binder ED 53 series
Silicon wafer Silicon Quest 708-007 Boron doped <100> wafer, 4" diameter, 500 μm, single side polished
GM1070 SU-8 photoresist Gersteltec Sarl
SU-8 developer Gersteltec Sarl
Sylgard 184 curing agent Dow Corning
Sylgard 184 elastomer prepolymer Dow Corning  
PLL-PEG-biotin (20%) SuSos AG PLL(20)-g[3.5]-PEG(2)/PEG(3.4)-Biotin (20%) 1 mg/ml in PBS
Fluorescein Sigma 46955 1 mM in PBS
Streptavidin-AlexaFluor350 Invitrogen S-11249 1 mg/ml in PBS
Biotin-4-fluorescein Invitrogen B-1370 0.03 μg/μl in PBS
Biotin-RGD GenScript SC1208 0.03 mg/ml in PBS
Syto 64 Red Invitrogen S-11346 1 μM in PBS
Sticky slide chemotaxis 3D Ibidi 80328
200 μl Greiner yellow bevelled tip Greiner Bio-One 739261
Vaseline Sigma 16415
Paraffin wax, mp 55-57 °C Sigma 327204
Nano eNabler 10 μm cantilever BioForce SPT-S-C-10s
Image J software National Institute of Health rsbweb.nih.gov/ij/download.html
Manual Tracking plugin Fabrice Cordelières rsb.info.nih.gov/ij/plugins/track/track.html
Chemotaxis and Migration Tool Ibidi GmbH www.ibidi.com/applications/ap_chemotaxis.html

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