The authors would like to thank: Dr. Frank Jiang, Dr. David Lin, Dr. Bernard Yurke and Dr. Uday Chippada for their contributions on developing the DNA gel technology; Dr. Norell Hadzimichalis, Smit Shah, Kimberly Peterman, Robert Arter for their comments and edits of this manuscript; funding sources including the New Jersey Commission on Spinal Cord Research (Grant #07A-019-SCR1, N.A.L.) and New Jersey Neuroscience Institute (M.L.P.); and publishers of Tissue Engineering, Part A for permission to reprint Figures 2 and 4 and Biomaterials for permission to reprint Figure 3.

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The authors have nothing to disclose.

The ability of DNA gels to soften or stiffen before and after cell adhesion makes them an ideal model to study the role of dynamic tissue stiffness on cell function. All three designs have been used in mechanical and biological studies. However, all three designs have similar elasticities at various crosslink percentages, indicating crosslink length does not influence DNA gel elasticity (Table 2). In contrast, acrylamide concentration affects elasticity. These designs may differ in crosslinking kinetics ...

Static and dynamic substrates are two categories of biomaterials that were developed to study the effects of tissue elasticity or stiffness on cell function. Static substrates are unable to change their physical properties after they are fabricated and/or once cells are plated. Polyacrylamide (PA) gels were the first two-dimensional, static substrates that were synthesized for mechanobiology investigations 5,17. PA gels are easy to prepare, inexpensive, versatile, and can be fabricated with a broad range of elastic moduli. Although these technical advantages make PA gels a commonly applied substrate, static substrates are not indicative of the dynamic nature of the extracellular matrix (ECM) and surrounding cellular environment in vivo. For example, the ECM undergoes stiffness alterations as a result of injury, development, or disease. Dynamic substrates are therefore favored as tissue-mimicking substrate models in mechanobiology studies 22,24,25.
Numerous synthetic, natural, two-dimensional, three-dimensional, static, and dynamic biomaterials have been developed to mimic tissue stiffness 1,3,6,16,23,26. Some dynamic substrates require heat, UV, electrical current, ions, and pH changes to alter their mechanical properties 2,4,7,8,12,15,16, but these stimuli can restrict the hydrogel&#8217;s bio-application. DNA-crosslinked polyacrylamide hydrogels (DNA gels) are dynamic two-dimensional elastic substrates. DNA crosslinks allow for temporal, spatial, and reversible modulation of DNA gel stiffness by addition of single-stranded DNA (ssDNA) to media or buffer 9-11,13,14,18,21. Unlike the aforementioned dynamic gels where stimuli are applied for modulation of elasticity, the DNA gels rely on the diffusion of applied ssDNA for the alteration of elasticity. Therefore, the upper gel surface, where cells are grown, is the first area modulated because the rate of elasticity modulation is dependent on the gel thickness.
DNA gels are similar to their PA gel counterparts in that they have a polyacrylamide backbone, however the bis-acrylamide crosslinks are replaced with crosslinks composed of DNA (Figure 1). Two ssDNAs (SA1 and SA2) hybridize with a crosslinker strand (L2) to make up the DNA crosslinks of the gel. SA1 and SA2 have distinct sequences that both contain an Acrydite modification at the 5&#180; end for effective incorporation into the PA network. For preparation of the gels, SA1 and SA2 are individually polymerized into a PA backbone and, subsequently, the polymerized SA1 and SA2 are mixed together. L2, the crosslinker, is added to the SA1 and SA2 mixture. The L2 base sequence is complementary to both SA1 and SA2 sequences and L2 hybridizes with SA1 plus SA2 to form the DNA crosslinks. Initial, DNA gel elasticity is determined by both L2 concentrations and crosslinking (Tables 1 and 2). DNA gels containing equal stoichiometric amounts of L2, SA1, and SA2 are the stiffest gels because SA1 and SA2 are 100% crosslinked by L2 (designated as 100% gels). Lower concentrations of L2 result in a lower percentage of DNA crosslinking and, therefore, softer DNA gels. Gels as low as 50% crosslinked (designated as 50% gels) have been constructed 9-11.
<img alt="Figure 1" src="/files/ftp_upload/51323/51323fig1highres.jpg" /> 
Figure 1.&#160;DNA gel crosslinking and uncrosslinking schematic 9-11,13,14,18,21.&#160; Step 1: SA1 (red) and SA2 (blue) are individually polymerized into a polyacrylamide backbone (black). After polymerization, SA1 and SA2 polymerized solutions are mixed together. Step 2: L2 (green) is added and hybridizes with SA1 plus SA2 to form the crosslinks of the gel. Step 3: R2 hybridizes with the toehold of L2. Step 4: Toehold hybridization of R2 propels the unzipping of L2 from SA1 and SA2.
Unlike PA gels, DNA gels can stiffen and soften after synthesis. For that reason, cells grown on DNA gels can be subjected to dynamic stiffness alterations. To stiffen cell-adherent gels, L2 can be added to the culture media of low percentage gels to increase the percentage of crosslinks. To soften cell-adherent gels, L2 can be removed to decrease the percentage of crosslinks 10,13,21. L2 has an additional toehold sequence at the 3&#180; end to allow L2 to uncrosslink from SA1 and SA2 (Table 1). Removal of L2 is accomplished by hybridization of a reversal strand called R2. R2 is complementary to the full length of L2 and hybridizes first with the L2 toehold. Toehold hybridization propels the unzipping of L2 from SA1 and SA2, which eliminates the crosslink and reduces the gel stiffness.
In this report and video, step-by-step instructions are provided for the preparation of stiffening and softening DNA gels. While 100% and 80% gel preparations are described, this protocol can be tailored to create DNA gels of other initial and final crosslinked percentages. In general, 100% and 80% gels are prepared, immobilized onto glass cover slips, functionalized, and seeded with cells. L2 is added to the media of 80% gels and R2 is added to the media of 100% gels, 48 hr after plating. The addition of L2 to media stiffens 80% gels to 100% crosslinked, whereas the addition of R2 to media softens 100% gels to 80% crosslinked. Stiffened gels are designated as 80&#8594;100% gels and softened gels are designated as 100&#8594;80% gels in the text. For control or static gels, ssDNA consisting of Ts or As is delivered to another set of 100% and 80% gels. After a minimum of two days following elasticity modulation, cells can be processed and analyzed.
<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always">
	<tbody align="center">
		<tr>
			<td rowspan="2"></td>
			<td rowspan="2">DNA crosslink</td>
			<td rowspan="2"># of bases</td>
			<td>Sequence (Toehold)</td>
			<td rowspan="2">Modification</td>
			<td rowspan="2">Melting temperature (Tm, &#176;C)</td>
			<td rowspan="2">Comments</td>
		</tr>
		<tr>
			<td>5&#39;&#8594;3&#39;</td>
		</tr>
		<tr>
			<td rowspan="4">Design 1</td>
			<td>SA1</td>
			<td>10</td>
			<td>GCA CCT TTG C</td>
			<td>5&#39; Acrydite</td>
			<td>34.9</td>
			<td></td>
		</tr>
		<tr>
			<td>SA2</td>
			<td>10</td>
			<td>GTC AGA ATG A</td>
			<td>5&#39; Acrydite</td>
			<td>23.6</td>
			<td></td>
		</tr>
		<tr>
			<td>L2</td>
			<td>30</td>
			<td>TCA TTC TGA CGC AAA GGT GCG CTA CAC TTG</td>
			<td></td>
			<td>56</td>
			<td>A 10 bp toehold sequence is included.&#160;</td>
		</tr>
		<tr>
			<td>R2</td>
			<td>30</td>
			<td>CAA GTG TAG CGC ACC TTT GCG TCA GAA TGA</td>
			<td></td>
			<td></td>
			<td>R2 is complementary to L2</td>
		</tr>
		<tr>
			<td rowspan="4">Design 2</td>
			<td>SA1</td>
			<td>14</td>
			<td>CGT GGC ATA GGA CT</td>
			<td>5&#39; Acrydite</td>
			<td>46.9</td>
			<td></td>
		</tr>
		<tr>
			<td>SA2</td>
			<td>14</td>
			<td>GTT TCC CAA TCA GA</td>
			<td>5&#39; Acrydite</td>
			<td>40.2</td>
			<td></td>
		</tr>
		<tr>
			<td>L2</td>
			<td>40</td>
			<td>TCT GAT TGG GAA ACA GTC CTA TGC CAC GGT TAC CTT CAT C</td>
			<td></td>
			<td>65.9</td>
			<td>A 12 bp sequence toehold is included.&#160;</td>
		</tr>
		<tr>
			<td>R2</td>
			<td>40</td>
			<td>GAT GAA GGT AAC CGT GGC ATA GGA CTG TTT CCC AAT CAG A</td>
			<td></td>
			<td>65.9</td>
			<td>R2 is complementary to L2</td>
		</tr>
		<tr>
			<td rowspan="3">Design 3</td>
			<td>SA1</td>
			<td>20</td>
			<td>ACG GAG GTG TAT GCA ATG TC</td>
			<td>5&#39; Acrydite</td>
			<td>55</td>
			<td></td>
		</tr>
		<tr>
			<td>SA2</td>
			<td>20</td>
			<td>CAT GCT TAG GGA CGA CTG GA</td>
			<td>5&#39; Acrydite</td>
			<td>56.6</td>
			<td></td>
		</tr>
		<tr>
			<td>L2</td>
			<td>40</td>
			<td>TCC AGT CGT CCC TAA GCA TGG ACA TTG CAT ACA CCT CCG T</td>
			<td></td>
			<td>68.8</td>
			<td>Toehold is not included.</td>
		</tr>
		<tr>
			<td rowspan="2">Control</td>
			<td rowspan="2">Control</td>
			<td rowspan="2">20-40</td>
			<td>AAA AAA (etc.) or</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>TTT TTT (etc.)</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;Base sequences for ssDNA 9-11,13,14,18,21. Cellular and mechanical studies have utilized several different crosslink designs to generate DNA gels with a range of static and dynamic mechanical properties. The parameters modulated in crosslink design are base sequence and sequence length or crosslink length. Bold and italicized fonts illustrate base pairing between SA1 and L2 and between SA2 and L2, respectively.
<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always">
	<tbody align="center">
		<tr>
			<td rowspan="2"></td>
			<td colspan="8">Design</td>
		</tr>
		<tr>
			<td colspan="3">1</td>
			<td colspan="3">2</td>
			<td colspan="2">3</td>
		</tr>
		<tr>
			<td>Acrylamide Concentration (%)</td>
			<td colspan="3">10</td>
			<td colspan="3">10</td>
			<td>10</td>
			<td>4</td>
		</tr>
		<tr>
			<td>SA1 plus SA2 hybridized to L2 (% crosslinked)</td>
			<td>50</td>
			<td>80</td>
			<td>100</td>
			<td>50</td>
			<td>80</td>
			<td>100</td>
			<td>100</td>
			<td>100</td>
		</tr>
		<tr>
			<td>Elasticity (kPa, Mean&#177;SEM)</td>
			<td>6.6 &#177; 0.6</td>
			<td>17.1 &#177; 0.8</td>
			<td>29.8 &#177; 2.5</td>
			<td>5.85&#177; 0.62</td>
			<td>12.67 &#177; 1.33</td>
			<td>22.88 &#177; 2.77</td>
			<td>25.2 &#177; 0.5</td>
			<td>10.4 &#177; 0.6</td>
		</tr>
	</tbody>
</table>
Table 2.&#160;Young&#8217;s modulus (E) of DNA gels 9-11,13,14,18,21. Acrylamide concentration, crosslink percentage, and crosslink length can be modulated in DNA gels. Designs 1, 2, and 3 have 20, 28, and 40 bp crosslink lengths, respectively. 100% gels for all designs have similar moduli indicating crosslink length does not affect gel elasticity. However, variations in acrylamide concentration alter DNA gel elasticity.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>ssDNA</td>
			<td>Integrated DNA Technologies (Coralville, Iowa) idtdna.com</td>
			<td></td>
			<td>Do not vortex ssDNA. Gentle invert the vial and/or pipette solution to mix.</td>
		</tr>
		<tr>
			<td>PBS with calcium and magnesium</td>
			<td></td>
			<td></td>
			<td>Any brand.</td>
		</tr>
		<tr>
			<td>100X Tris-EDTA buffer (TE buffer)</td>
			<td>Sigma-Aldrich (St. Loius, MO) sigmaldrich.com</td>
			<td>T9285</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Tris-Borate-EDTA buffer (TBE buffer)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>93290</td>
			<td>TBE is a reproductive toxin.</td>
		</tr>
		<tr>
			<td>40% Acrylamide solution</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>BP14021</td>
			<td>Acrylamide is a toxin.</td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>A3678</td>
			<td>Prepare a 2% solution in TE buffer. APS is a toxin and irratant.</td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>T9281</td>
			<td>Prepare a 20% solution in TE buffer. TEMED is flammable, a corrosive, and a toxin.</td>
		</tr>
		<tr>
			<td rowspan="2">12-mm diameter round coverglass</td>
			<td rowspan="2">Fisher Scientific (Pittsburg, PA) fishersci.com</td>
			<td rowspan="2">12-545-82</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Norland optical adhesive 72</td>
			<td>Norland Products (Cranbury, NJ) norlandprod.com</td>
			<td>NOA72</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">24-well tissue culture plate</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">Any brand.</td>
		</tr>
		<tr>
			<td rowspan="2">Microcentrifuge tubes</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">Any brand.</td>
		</tr>
		<tr>
			<td>Sulfo-SANPAH</td>
			<td>ProteoChem or Thermo Fisher, (Rockland, IL) proteochem.com or thermofisher.com</td>
			<td>C111 or 22589</td>
			<td>Prepare a 0.315 mg/ml solution in water immediately before use. Dissolve at 37&#176;C and filter sterilze. It is normal to observe undisolved sulfo-SANPAH in the filter. Sulfo-SANPAH is light sensitive and, therefore, the solution should be protect from light until UV exposure.</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine (PDL)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>P6407</td>
			<td>Prepare a 0.2 mg/ml solution in water and filter sterilize.</td>
		</tr>
		<tr>
			<td>Collagen Type I</td>
			<td>Affymetrix (Santa Clara, CA) affymetrix.com</td>
			<td>13813</td>
			<td>Prepare a 0.2 mg/ml solution in 0.2 N acetic acid. Solution needs to remain cold at all times to avoid polymerization. Acetic acid is a flammable, toxic, and corrosive.</td>
		</tr>
		<tr>
			<td>22 X 60 cover glass</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>12-544-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive-displacement pipette</td>
			<td>Gilson, Inc (Middletown, WI) gilson.com</td>
			<td>F148504</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat block</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>11-718</td>
			<td></td>
		</tr>
		<tr>
			<td>UV light source</td>
			<td></td>
			<td></td>
			<td>Place gels as close as possible to the UV light. UV light can cause skin or eye injury.</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td></td>
			<td></td>
			<td>Any brand.</td>
		</tr>
		<tr>
			<td>Nitrogen gas</td>
			<td>GTS-Welco (Flemington, NJ) www.praxairmidatlantic.com/</td>
			<td>NI 5.0UH-R</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of dna-crosslinked polyacrylamide hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Prior to our studies, cell-ECM interactions were observed on static compliant biomaterials or on irreversible and unidirectional dynamic substrates. These substrates do not accurately reflect the dynamic nature of the cellular microenvironment. Our work shifts the existing technical paradigms by providing a more physiologic model for studying cell-ECM interactions on softening and stiffening dynamic biomaterials. In previous studies, we analyzed the effects of dynamic substrates on cells by examining various cell morphol...

Watch this Scientific Journal Video about Preparation of DNA-crosslinked Polyacrylamide Hydrogels at JoVE.com

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

preparation-of-dna-crosslinked-polyacrylamide-hydrogels

Research

JoVE Journal

Biology

14.5K Views.  JFK Medical Center. Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

Video: Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Horizontal Slice Preparation of the Retina

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of retinal neurons, such as amacrine cells, expand their dendrites horizontally, so that the morphology of the cells is supposed to be severely damaged in the vertical slices. In the whole-mount preparation, especially for patch-clamp recordings, retinal neurons in the middle layer are not easily accessible due to the extensive coverage of glial cell (Mueller cell) s endfeets. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact. The slice was made horizontally at the inner layer of the retina using a vibratome slicer after the retina was embedded in the low-temperature melting agarose gel. In this horizontal slice preparation of the retina, we studied the function of retinal neurons compared with their morphology, by using patch-clamp recording, calcium imaging technique, immunocytochemistry, and single-cell RT-PCR.

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact.

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of ...

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.
Several improvements of the original Coomassie protocol1 have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution2, and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol3. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol4. The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.
Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.

This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and ...

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 &deg;C during the gel run allows for the separation of unstructured DNA or RNA molecules.
In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.
In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol 1,2.

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Preparation of Quality Inositol Pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells are inositol pentakisphosphate (IP5) and inositol hexakisphosphate (phytic acid or IP6). IP5 and IP6 are the precursors of inositol pyrophosphate molecules that contain one or more pyrophosphate bonds1. Phosphorylation of IP6 generates diphoshoinositolpentakisphosphate (IP7 or PP-IP5) and bisdiphoshoinositoltetrakisphosphate (IP8 or (PP)2-IP4). Inositol pyrophosphates have been isolated from all eukaryotic organisms so far studied. In addition, the two distinct classes of enzymes responsible for inositol pyrophosphate synthesis are highly conserved throughout evolution2-4.
The IP6 kinases (IP6Ks) posses an enormous catalytic flexibility, converting IP5 and IP6 to PP-IP4 and IP7 respectively and subsequently, by using these products as substrates, promote the generation of more complex molecules5,6. Recently, a second class of pyrophosphate generating enzymes was identified in the form of the yeast protein VIP1 (also referred as PP-IP5K), which is able to convert IP6 to IP7 and IP87,8.
Inositol pyrophosphates regulate many disparate cellular processes such as insulin secretion9, telomere length10,11, chemotaxis12, vesicular trafficking13, phosphate homeostasis14 and HIV-1 gag release15. Two mechanisms of actions have been proposed for this class of molecules. They can affect cellular function by allosterically interacting with specific proteins like AKT16. Alternatively, the pyrophosphate group can donate a phosphate to pre-phosphorylated proteins17. The enormous potential of this research field is hampered by the absence of a commercial source of inositol pyrophosphates, which is preventing many scientists from studying these molecules and this new post-translational modification. The methods currently available to isolate inositol pyrophosphates require sophisticated chromatographic apparatus18,19. These procedures use acidic conditions that might lead to inositol pyrophosphate degradation20 and thus to poor recovery. Furthermore, the cumbersome post-column desalting procedures restrict their use to specialized laboratories.
In this study we describe an undemanding method for the generation, isolation and purification of the products of the IP6-kinase and PP-IP5-kinases reactions. This method was possible by the ability of polyacrylamide gel electrophoresis (PAGE) to resolve highly phosphorylated inositol polyphosphates20. Following IP6K1 and PP-IP5K enzymatic reactions using IP6 as the substrate, PAGE was used to separate the generated inositol pyrophosphates that were subsequently eluted in water.

Inositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates.

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...