Name: Microcontact Printing of Proteins for Cell Biology (Video) | JoVE Journal
Uploaded: 2008-12-05T00:00:00.000+00:00
Duration: 9 min 21 s
Description: Learn microcontact printing to pattern fibronectin on glass surfaces for studying cell adhesion. This technique enables researchers to recreate complex extracellular environments in controlled conditions.

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5 minutes

1. Preparation of solutions and materials
These steps should be carried out several days in advance.
<ol>
	<li>Glass coverslips. Coverslips were cleaned by immersion for 10 minutes into a solution of Linbro 7X detergent : water, mixed at a 1 : 3 ratio and heated, with stirring, until clear. Coverslips were rinsed extensively with deionized water, and then baked at 450°C for 6 hours. Loading coverslips into ceramic staining racks (see Reagents) simplified this process.</li>
	<li>Protein solution for stamping. Reconstitute fibronectin following the manufacturers instructions to a stock solution of 1 mg/ml concentration.</li>
</ol>
2. Cast stamps from the topological master
These steps can be carried out several days in advance. Store stamps pattern-up in a covered dish, such as a tissue culture dish.
<ol>
	<li>Remove loose dust from master using a stream of compressed, filtered air or inert gas.</li>
	<li>Place the master, patterned side up, in the bottom of a plastic dish just larger than the master. 60- or 100-mm tissue culture dishes are well suited for this purpose.</li>
	<li>In a polystyrene 50 ml centrifuge tube, combine the Sylgard components at a ratio of curing agent : elastomer base of 1 : 10 by weight. Mix thoroughly using a plastic device, such as a disposable pipette. Prepare at least 0.2 ml of elastomer per square centimeter of dish area.</li>
	<li>Centrifuge the 50 ml tube at 300 G for 5 min to remove air bubbles.</li>
	<li>Pour the elastomer over the master, then place in a desiccator, under vacuum, for 30 minutes.</li>
	<li>Cure the elastomer in an oven at 65°C for at least 2 hours. Curing at higher temperatures and for longer times results in stiffer elastomer. Let cool to room temperature.</li>
	<li>Separate the sheet of stamps from the master.</li>
</ol>
3. Microcontact printing of fibronectin onto glass
<ol>
	<li>Cut out a single stamp. Stamps measuring 4 mm X 4 mm to 1 cm X 1 cm in area and 1 – 2 mm thick are easiest to start with. Place pattern side up on a glass slide or small plastic dish.</li>
	<li>Place stamp in a plasma cleaner, and process, under vacuum, for 30 seconds. A Harrick Scientific plasma cleaner (see Equipment), set at its highest output setting will render the PDMS surface hydrophilic. Longer times result in cracking of the elastomer.</li>
	<li>Dilute the fibronectin solution with deionized water to a stamping concentration of 50 µg/ml.</li>
	<li>Place a small drop (10 – 50 µl) of stamping solution on the stamp. It will spread across the hydrophilic surface. Add only enough solution that the drop covers the stamp, but does not run over the edges. Let protein adsorb to stamp for 5 minutes.</li>
	<li>Using a Kimwipe® or other clean paper tissue, wick off most of the protein solution from the stamp, without touching the patterned region.</li>
	<li>Dry the remaining solution from the stamp under a stream of clean, dry, inert gas, such as nitrogen.</li>
	<li>Using tweezers, remove the stamp from the glass slide, invert, and place in contact with the cleaned glass coverslip (the surface to be patterned). Place a weight on top to promote good contact. The specific weight that provides the best patterning is dependent on the stamp size and pattern; start with a 5 g weight, and adjust between stampings. Leave stamp in contact with surface for 1 minutes.</li>
	<li>Carefully disassemble the stack, and separate stamp from coverslip.</li>
	<li>Vigorously rinse the patterned coverslip in PBS, followed by deionized water, to remove protein that is not adsorbed to the surface. Dry coverslip under a nitrogen stream.</li>
</ol>
 Representative Results
Microcontact printing is a powerful process for patterning molecules on surfaces. This process has the ability to create features with dimensions ranging from tens of micrometers to hundreds of nanometers; in Fig. 1A, the logos on the left are each 200 µm in height, while the green spots illustrated in Fig. 1B are 1 µm in diameter and spaced at 4 µm intervals, measured center-to-center. Fig. 1B also illustrates a powerful property of microcontact printing, namely that it is and additive process. Multiple rounds of microcontact printing can be applied to a single surface to create multicomponent systems3.
<img alt="Fluorescence microscopy diagram; protein expression analysis; visualized with optical markers." src="/files/ftp_upload/old/1065_2008_11_24_3/figure_a.jpg" data-altupdatedat="1787630408000" data-alt="Figure A"> <img alt="Photonic crystal diagram with green and red dot pattern for optical emission analysis." src="/files/ftp_upload/old/1065_2008_11_24_4/FIGURE_B.jpg" data-altupdatedat="1787630408000" data-alt="Figure B">

<ol><li>Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Geometric+control+of+cell+life+and+death%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Geometric control of cell life and death.</a> Science. 276, 1425-1425 (1997).</li>
<li>Kumar, A., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Features+of+Gold+Having+Micrometer+to+Centimeter+Dimensions+can+be+Formed+Through+a+Combination+of+Stamping+with+an+Elastomeric+Stamp+and+an+Alkanethiol+%22Ink%22+Followed+by+Chemical+Etching%5BTitle%5D)+AND+%22Applied+Physics+Letters%22%5BJournal%5D)">Features of Gold Having Micrometer to Centimeter Dimensions can be Formed Through a Combination of Stamping with an Elastomeric Stamp and an Alkanethiol "Ink" Followed by Chemical Etching.</a> Applied Physics Letters. 63, 4-4 (1993).</li>
<li>St. John, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preferential+Glial+Cell+Attachment+to+Microcontact-printed+Surfaces%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">Preferential Glial Cell Attachment to Microcontact-printed Surfaces.</a> Journal of Neuroscience Methods. 75, 171-171 (1997).</li>
<li>Kam, L., Boxer, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+adhesion+to+protein-micropatterned-supported+lipid+bilayer+membranes%5BTitle%5D)+AND+%22Journal+of+Biomedical+Materials+Research%22%5BJournal%5D)">Cell adhesion to protein-micropatterned-supported lipid bilayer membranes.</a> Journal of Biomedical Materials Research. 55, 487-487 (2001).</li>
<li>Kung, L. A., Kam, L., Hovis, J. S., Boxer, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patterning+Hybrid+Surfaces+of+Proteins+and+Supported+Lipid+Bilayers%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">Patterning Hybrid Surfaces of Proteins and Supported Lipid Bilayers.</a> Langmuir. 16, 6773-6773 (2000).</li>
<li>Shen, K., Thomas, V. K., Dustin, M. L., Kam, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Micropatterning+of+costimulatory+ligands+enhances+CD4%2B+T+cell+function%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Micropatterning of costimulatory ligands enhances CD4+ T cell function.</a> Proceedings of the National Academy of Sciences. 105, 7791-7791 (2008).</li>
<li>Shi, P., Shen, K., Kam, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Local+presentation+of+L1+and+N-cadherin+in+multicomponent%2C+microscale+patterns+differentially+direct+neuron+function+in+vitro%5BTitle%5D)+AND+%22Developmental+Neurobiology%22%5BJournal%5D)">Local presentation of L1 and N-cadherin in multicomponent, microscale patterns differentially direct neuron function in vitro.</a> Developmental Neurobiology. 67, 1765-1765 (2007).</li>
</ol>

The authors have nothing to disclose.

The microcontact printing process is conceptually simple and very robust, having been applied to patterning a wide range of molecules on a variety of substrates. However, this process remains something of an art. The specific geometry of the pattern to be created, protein to be patterned, applied weight, and coating/stamping conditions all affect the stamping quality. For example, too little weight, applied to large features, often results in gaps in the pattern as can be seen in the upper right logo of Fig. 1A. Converse...

<table><tbody><tr><td>Plasma Cleaner</td><td></td><td>Harrick Scientific Products, Inc.</td><td>PDC-32G</td><td></td></tr><tr><td>Desiccator</td><td></td><td>Nalge Nunc international</td><td>5315-0150</td><td></td></tr><tr><td>PBS</td><td>Reagent</td><td>Invitrogen</td><td>10010-072</td><td></td></tr><tr><td>Protein labeling kit</td><td>Reagent</td><td>Invitrogen</td><td>A30006</td><td></td></tr><tr><td>Fibronectin </td><td>Reagent</td><td>Sigma-Aldrich</td><td>F2006</td><td></td></tr><tr><td>Staining rack</td><td>Reagent</td><td>Thomas Scientific</td><td>8542E40</td><td></td></tr><tr><td>Coverslips</td><td>Reagent</td><td>Fisher Scientific</td><td>12-544-12</td><td></td></tr><tr><td>Sylgard 184</td><td>Reagent</td><td>Ellsworth Adhesives</td><td>184 Sil Elast Kit</td><td></td></tr><tr><td>Diffraction Grating</td><td>Reagent</td><td>Edmund Scientific</td><td>3040267</td><td></td></tr></tbody></table>

microcontact printing of proteins for cell biology

Microcontact Printing of Proteins for Cell Biology

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. Development of microcontact printing by the Whitesides group (<a href="http://gmwgroup.harvard.edu/">http://gmwgroup.harvard.edu/</a>) in the mid-1990s revolutionalized this field by making microelectronics/microfabrication techniques accessible to laboratories focused on the life sciences. Initial implementations of this method used polydimethylsiloxane (PDMS) stamps to create patterns of functionalized chemicals on material surfaces1. Since then, a range of innovative approaches have been developed to pattern other molecules, including proteins2. This video demonstrates the basic process of creating PDMS stamps and uses them to pattern proteins, as these steps are difficult to accurately express in words. We focus on patterning the extracellular matrix protein fibronectin onto glass coverslips as a specific example of patterning.
An important component of the microcontact printing process is a topological master, from which the stamps are cast; the raised and lowered regions of the master are mirrored into the stamp and define the final pattern. Typically, a master consists of a silicon wafer coated with photoresist and then patterned by photolithography, as is done here. Creation of masters containing a specific pattern requires specialized equipment, and is best approached in consultation with a fabrication center or facility. However, almost any substrate with topology can be used as a master, such as plastic diffraction gratings (see Reagents for one example), and such serendipitous masters provide readily available, simple patterns. This protocol begins at the point of having a master in hand.

Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces. We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass.

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. ...

Micro contact printing is used to pattern proteins and other molecules on material surfaces to recreate the complexity of extracellular environments. To begin micro contact printing, a topological master is created and then soft lithography is used to cast a stamp from the master out of Syl guard. The stamp can be coated with protein and then used to stamp proteins on a new surface on which cells can adhere.Hi, my name is Chen. I'm from Dr.Lance Camp's lab in biomedical engineering Columbia University. Hi, I'm GT from the Mechanical Engineering Department.Hello, I'm Lance Kem from the Department of Biomedical Engineering at Columbia University. We use this procedure in our laboratory to study how cells respond to complex arrangements of proteins in their extracellular environment. This was developed in the mid 1990s by the laboratory of George Whitesides and has become a widely used and valuable tool.There are many variations to this technique, but today G and Co U will be demonstrating the basic micro contact printing process. So let's get started. For micro contact printing, you should prepare the cover slips and protein solution for stamping several days in advance.First, immerse the cover slips into the limbo solution for 10 minutes heating and stirring until the solution is clear. This process as well as the following steps for cleaning and storage can be simplified by loading cover slips into ceramic staining racks. Next extensively, rinse the cover slips with deionized water bake the rinse cover slips at 45 degrees Celsius for six hours.To prepare the protein solution, reconstitute fibronectin following the manufacturer's instructions to a stock of one milligram per milliliter concentration. These stocks are stored frozen at negative 20 degrees Celsius. With working aliquots in temporary storage at four degrees Celsius.Casting stamps can be carried out several days in advance of the experiment. These stamps can be stored pattern up in a covered dish like a tissue culture dish. To begin, first, remove loose dust from the master using a stream of compressed filtered air or inert gas.Next, place the master pattern side up in the bottom of a plastic dish, which is slightly larger than the master and should be coated in aluminum foil. 60 or 100 millimeter tissue culture dishes work well for this purpose. In the polystyrene 50 milliliter centrifuge tube, combine the cyl guard components at a ratio of curing agent to elastomer base of one to 10 by weight.Mix thoroughly with a disposable plastic instrument like a pipette tip. Prepare enough of the elastomer such that there is at least 0.2 milliliters of elastomer per square centimeter of dish area. Centrifuge the 50 milliliter tube at 300 G for five minutes.To remove air bubbles after centrifugation or the elastomer over the master, then place under vacuum such as inside a desiccate for 30 minutes. Next, cure the elastomer in an oven at 65 degrees Celsius for at least two hours. Curing at higher temperatures and for longer times results in stiffer elastomer.After curing, let the master cool to room temperature. Finally, separate the sheet of stamps from the master and then trim stamp to the dimensions of the master to micro contact. Print fiber nin onto the glass cover slips first.Cut out a single stamp stamps measuring four millimeter by four millimeter to one centimeter by one centimeter in area and one to two millimeter thick are easiest to start with. Place the stamp pattern side up on a glass slide or small plastic dish. Next, place the stamp in a plasma cleaner and process under a vacuum for 30 seconds.A herrick scientific plasma cleaner set at its highest output setting will render the PDMS surface hydrophilic. If this is set for longer times, it will result in cracking of the elastomer. Dilute the fiber nin solution with PBS to a stamping concentration of 50 micrograms per milliliter.Once the solution is ready, place a small drop of about 10 to 50 microliters of the stamping solution on the stamp. The solution will spread across the hydrophilic surface. Add only enough solutions so that the drop covers the stamp, but does not run over the edges.Let protein absorb to the stamp for five minutes. Afterwards, use a Kim wipe or other clean paper tissue to wick off most of the protein solution from the stamp without touching the patterned region. Dry the remaining solution from the stamp under a stream of clean, dry inert gas such as nitrogen.Once dry, use tweezers to remove the stamp from the glass slide invert and place the stamp in contact with the cleaned glass cover. Slip the surface to be patterned. Place a weight on top of the stamp to promote good contact.The specific weight that provides the best patterning is dependent on the stamp size and pattern. Start with the five gram weight and adjust between stampings. Leave the stamp in contact with the surface for one minute.Once finished, carefully disassemble the stack and separate the stamp from the cover Slip vigorously. Rinse the patterned cover slip in PBS followed by deionized water to remove protein that is not absorbed to the surface dry the cover slip under a nitrogen stream. Micro contact printing is a powerful process for creating patterns of molecules on surfaces.In this example, we pattern fluorescently labeled fibronectin in the pattern of our school logo. Each logo measures 200 micrometers in height. A powerful aspect of this process is that multiple rounds of micro contact printing can be combined on a single surface, as in this example of two different antibodies overlaid on a single surface.In this case, the green spots measure one micrometer in diameter and are spaced at four micrometer intervals. We use these surfaces to investigate how cells integrate multiple cues of the extracellular environment. In this movie, naive T cells are seated onto an array of spots containing antibodies to cell surface receptors.Cells locate and interact with these features, self-organizing irregular arrays. The micro contact printing process is conceptually simple and very robust, having been applied to patterning a wide range of molecules on a variety of substrates. However, this process remains something of an art.The specific geometry of the pattern to be created, protein to be patterned applied weight and coating stamping conditions all affect the stamping quality. We demonstrate the patterning of fibronectin on the glass as a reliable starting point for learning these techniques. We have just shown you the basics of micro contact stamping when doing this procedure.Always remember to follow the lab safety precautions. So that's it. Thank You for watching and good luck with your experiments.

microcontact-printing-of-proteins-for-cell-biology

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JoVE Journal

Biology

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Learn microcontact printing to pattern fibronectin on glass surfaces for studying cell adhesion. This technique enables researchers to recreate complex extracellular environments in controlled conditions.

Microcontact Printing of Proteins for Cell Biology

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