This work was funded by start-up funds provided to Dr. Silviya Zustiak by Saint Louis University as well as by a President&#8217;s Research Fund (PRF) grant awarded to Dr. Silviya Zustiak by Saint Louis University. We thank Naveed Ahmed and Keval Shah for technical assistance.

1. Preparation of Hydrogel-associated Solutions and Aliquots
<ol>
	<li>Preparation of polyacrylamide gel precursor solution.
	<ol>
		<li>Prepare polyacrylamide gel precursor solution by mixing acrylamide (A) (40% w/v, Mr 71.08 g/mol), the crosslinker bisacrylamide (B) (2% w/v, Mr 154.17 g/mol), and de-ionized water in the volume percentages specified in Table 1. 
		NOTE: These solutions can be prepared in large batches and stored at 4 &#176;C for up to several months.
		<ol>
			<li>CAUTION: Acrylamide is toxic upon inhalation or ingestion, particularly, when in powder form: thus, preferably use 40% w/v solution to reduce toxicity risks. Handle only while wearing protective clothing, such as gloves, goggles, and a lab coat. Store in a light-resistant, tightly closed container in the fridge (&#60;23 &#176;C, well-ventilated area).</li>
			<li>CAUTION: Bis-acrylamide is toxic upon inhalation or ingestion, particularly, when in powder form: thus, preferably use 2% w/v solution to reduce toxicity risks. Handle only while wearing protective clothing, such as gloves, goggles, and a lab coat. Store in a light-resistant, tightly closed container in the fridge (&#60;4 &#176;C, well-ventilated area).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation and usage of N-Sulfosuccinimidyl-6-(4&#39;-azido-2&#39;-nitrophenylamino) hexanoate (Sulfo-SANPAH, Mr 492.40 g/mol) aliquots. CAUTION: Sulfo-SANPAH causes serious eye irritation; handle with gloves and proper eye or face protection. Store at -20 &#176;C upon receipt and prior to aliquoting.
	<ol>
		<li>To store sulfo-SANPAH: dissolve Sulfo-SANPAH in dimethylsulfoxane (DMSO) at 50 mg/ml. Aliquot the stock solution into 50 micro centrifuge tubes of 20 &#956;l per tube. Flash freeze on dry ice or in liquid nitrogen (an optional but preferred step to preserve maximum crosslinker efficiency) and store at -80 &#176;C. 
		NOTE: The aliquots can be stored for several months.</li>
		<li>To use sulfo-SANPAH: thaw the aliquot briefly and dilute in 480 &#956;l of de-ionized water. Use immediately. Sulfo-SANPAH hydrolyzes quickly in water: therefore take caution to perform all of the above steps quickly.</li>
	</ol>
	</li>
	<li>Preparation of ammonium persulfate (Mr 228.18 g/mol) aliquots. 
	NOTE: Ammonium persulfate causes eye, skin and respiratory irritation. Wear appropriate personal protective equipment when handling. Handle ammonium persulfate powder in a chemical fume hood. Store powder in a dry, well-ventilated place. Once aliquoted, the dilute solution could be used on the work bench.
	<ol>
		<li>Dissolve ammonium persulfate in deionized water to achieve a final ammonium persulfate concentration of 10% w/v. Aliquot and store at -20 &#176;C. Thaw immediately prior to use.</li>
	</ol>
	</li>
	<li>Preparation of Collagen Type I solution.
	<ol>
		<li>Prepare 0.2 mg/ml collagen solution by diluting the stock solution in 1x phosphate buffered saline (PBS) of pH 7.4. Store on ice briefly or use immediately upon dilution.</li>
	</ol>
	</li>
</ol>
2. Hydrogel Preparation (Refer to Figure 1)
<ol>
	<li>Preparation of hydrophobic glass slides.
	<ol>
		<li>Place a few drops of a hydrophobic solution on a glass plate and use tissue paper to spread across the surface. Let air dry and wipe with a tissue paper again to even out the hydrophobic coating. 
		NOTE: Store hydrophobic solution in a flammable cabinet at RT. Wear personal protective equipment when handling. Work in a well-ventilated area.</li>
	</ol>
	</li>
	<li>Preparation of flexible plastic support.
	<ol>
		<li>Cut the flexible plastic support to match the size of the hydrophobic-coated glass plate.</li>
		<li>Mark the hydrophobic side of the flexible plastic support by lightly scratching the surface with a sharp tool such as a scalpel. 
		NOTE: Once the gel &#8212; which is fully transparent &#8212; dries, the scratch marks will help distinguish which flexible plastic support side contains the gel. 
		NOTE: The flexible plastic support has a hydrophobic and a hydrophilic side. Once deposited, polyacrylamide permanently adheres to the hydrophilic side of the plastic support which facilitates easy subsequent hydrogel handling.</li>
	</ol>
	</li>
	<li>Gel Preparation (example volumes are given for 5 ml gel precursor solutions). 
	NOTE: 5 ml would be sufficient to produce ~40 gels when the gel initial thickness is 0.5 mm. More gels can be produced if the gel thickness is reduced.
	<ol>
		<li>Place 4972.5 &#956;l of polyacrylamide precursor solution of desired final concentration (Table 1) into a 50 ml conical tube. Place in a degassing chamber for 30 min with the cap opened. 
		NOTE: Oxygen acts as a free radical trap and when present, it inhibits polymerization.</li>
		<li>Add 25 &#181;l of 10% w/v ammonium persulfate (refer to point 1.3) to achieve a final ammonium persulfate concentration of 0.05%.</li>
		<li>Add 2.5 &#181;l of N,N,N&#8217;,N&#8217;-tetramethylethylenediamine (TEMED, Mr 116.24 g/mol) to the degassed gel solution to achieve a final TEMED concentration of 0.5%. 
		NOTE: Store TEMED in a flammable cabinet at RT. Handle in a chemical fume hood when wearing appropriate personal protective equipment. Keep tightly closed under inert gas, as it is highly air and moisture sensitive.</li>
		<li>Mix the solution gently by pipetting up and down 3 &#8211; 5 times. Do not vortex to avoid oxygen diffusion into the gel precursor solution.</li>
		<li>Pipette the gel solution onto the hydrophilic side of the flexible plastic support and sandwich with hydrophobic-coated glass slide. Separate the two slides with silicone spacers of desired thickness (e.g., 0.5 mm). Leave a small amount (~100 &#956;l) of polymer precursor solution in the 50 ml conical tube to use as an indicator of gelation. 
		NOTE: Any spacer could be used. For example, a single strip of Parafilm gives a final swollen gel thickness of 100 &#8211; 120 &#956;m.</li>
		<li>Place another glass plate atop the flexible plastic support/gel sandwich to ascertain an even hydrogel surface upon polymerization.</li>
		<li>Let the gel polymerize for 45 min, although less time can be used for gels of higher % wt if desired. To ascertain that the gel has polymerized, observe the remaining solution in the 50 ml conical tube; if the remaining solution has gelled, then it is likely that gelation on the glass plate has been initiated as well. However, note that premature opening of the mold will prevent complete polymerization.</li>
		<li>Once the gel is formed, peel off the flexible plastic support with the covalently attached polyacrylamide gel on top, and set gel-side-up to air dry. 
		NOTE: Convection or heat drying can also be used to accelerate this step. Once dried onto the flexible plastic support, the gel can be stored indefinitely.
		<ol>
			<li>Mark any bare spots of the flexible plastic support, where polyacrylamide gels did not form due to bubbles. Mark while the gel is still hydrated as this will blend in with the flexible plastic support once the gel is dry.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Multiwell Plate Assembly, Collagen Coating, and Sterilization
<ol>
	<li>Multiwell plate assembly.
	<ol>
		<li>Once dried, cut PA gels into desired shapes.
		<ol>
			<li>For a 96-well plate, use a heavy-duty hole punch with a diameter of ~6 mm. Use a heavy-duty paper cutter to cut gels into square or rectangular shapes. Alternatively, use scissors.</li>
		</ol>
		</li>
		<li>Prepare approximately 500 &#956;l of PDMS per 96-well plate according to the manufacturer&#8217;s instructions. To glue the gels to the bottom of a multiwell plate, place a small droplet (~5 &#956;l) of polydimethylsiloxane (PDMS) at the center of the each well. Using forceps, place one polyacrylamide gel in each well, flexible plastic support side down. To allow PDMS to cure, leave the assembled plate at 37 &#176;C for a minimum of 4 hr.</li>
	</ol>
	</li>
	<li>Collagen coating of polyacrylamide gels.
	<ol>
		<li>With a transfer pipette, place a small amount (7 &#8211; 8 &#956;l) of sulfo-SANPAH (refer to point 1.2.2) in each well and swirl from side to side to coat the gel surface evenly. Work promptly since sulfo-SANPAH is not stable in water.</li>
		<li>Place the well plate under a high intensity UV lamp (intensity = 37 mW, &#955; = 302 &#8211; 365 nm) for 5 min. Rinse the gels with PBS to remove excess sulfo-SANPAH.</li>
		<li>Pipette 50 &#181;l of 0.2 mg/ml Collagen Type I solution (refer to point 1.4) into each well. Leave the plate covered at RT for at least 2 hr or O/N at 4 &#176;C. 
		NOTE: To speed up the process of collagen coating, a transfer pipette can be used to add the collagen solution. Typically, 1 &#8211; 2 droplets of solution should be enough to completely cover the gel surface.</li>
		<li>Leave the well plate at RT for at least 2 hr to allow collagen bonding.</li>
		<li>Rinse with PBS to remove excess collagen solution and sterilize under UV (&#955; = 200 nm) in a tissue culture hood for 2 hr.</li>
		<li>Soak the gels in complete medium (refer to section 4.1 for medium composition) O/N to hydrate and equilibrate. Use for cell seeding immediately or store in the fridge for up to 2 days.</li>
	</ol>
	</li>
</ol>
4. Cell Seeding on PA Stiffness Assay
NOTE: Although typical for common mammalian cell lines, the protocol outlined in this section is specifically used with the breast cancer MDA-MB-231 cell line (see Figures 4 and 5).
<ol>
	<li>Collect cells from tissue culture flask by exposure to 5% trypsin/EDTA for 5 min at 37 &#176;C. Use ~80 &#956;l of trypsin/EDTA per each cm2 of culture flask area; for example, use 2 ml of trypsin/EDTA for a T25 cell culture flask. Re-suspend collected cells in complete medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at a desired final cell concentration. Taking into account cell doubling time as well as the length of time the cells will be seeded on the assay, choose an appropriate sub-confluent cell concentration. Ensure that added medium is enough to completely submerge the hydrogels. 
	NOTE: Since the hydrogels have been pre-equilibrated in media (refer to step 3.2.6) 100 &#956;l volume should be sufficient. Typical medium volumes used for multiwell plates should be sufficient since the gels have been pre-equilibrated in media in the previous step. 
	NOTE: The assay would be appropriate for any attachment-dependent cell type.
	<ol>
		<li>Count cell number under an inverted microscope by using a hemacytometer. Load 10 &#181;l of cell suspension into each hemacytometer port and average the cell count from at least 8 quadrants. To get the final cell concentration, multiply the cell count by 104. 
		NOTE: Best cell count results are obtained when cell number in each hemacytometer quadrant is 20 &#8211; 50.</li>
	</ol>
	</li>
	<li>Culture the cells onto the PA stiffness assay in a humidified incubator at 37 &#176;C and 5% CO2. Change media every 2 &#8211; 3 days. Collect cells when needed by exposure to 5% trypsin/EDTA at 37 &#176;C for 5 min. Use ~80 &#956;l of trypsin/EDTA per cm2 of tissue culture flask. During all cell manipulation steps, take special care not to disrupt the hydrogel surface: aspirate or pipette medium by slightly tipping the multiwell plate sideways and touching the pipette tip onto the side wall of each well, as opposed to touching the hydrogel. 
	NOTE: Except for taking special care not to damage the hydrogel surface during standard tissue culture handling, the cells seeded onto the stiffness assay can be manipulated the same way as if they were seeded onto a regular multiwell plate.</li>
</ol>
5. Imaging of Cells Seeded onto PA Stiffness Assay
<ol>
	<li>Image cells directly on the PA stiffness assay. 
	NOTE: Any microscope &#8212; inverted, fluorescent, or confocal, can be used for cell imaging. 
	NOTE: The flexible plastic support used to construct the stiffness assay is fully transparent and does not autofluoresce or interfere with cell imaging. However, even though the flexible plastic support itself is transparent, imaging capabilities will be limited by the working distance of the objective. The flexible plastic support has a thickness of 0.23 mm and a typical working distance of a 10X objective is ~4 mm, sharply decreasing for higher magnifications.
	<ol>
		<li>For live cell imaging, position PA stiffness assay in microscope plate holder and image. Keep imaging sessions under 2 hr, or use a microscope equipped with an environmental chamber for longer imaging times.</li>
		<li>When live cell imaging is not the goal, fix cells in 4% formaldehyde solution supplemented with 0.1% detergent. Soak cells in fixative at RT for a minimum of 2 hr. Rinse with PBS twice. Discard fixative waste in a designated waste container. 
		NOTE: Cells can be imaged immediately or stored submerged in PBS at 4 &#176;C for up to 2 weeks. CAUTION: Formaldehyde is toxic upon inhalation and contact. Handle with gloves in a chemical fume hood. 
		NOTE: Cell fixation protocol has to be chosen based on the subsequent cell staining and handling protocols, because certain fixatives damage some cell proteins.</li>
		<li>For fluorescent imaging, stain fixed cells with desired cell stain directly in the multiwell plate. Image immediately.</li>
	</ol>
	</li>
</ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+rapid+method+for+generating+patterned+co-cultures+with+stable+interfaces.">A simple and rapid method for generating patterned co-cultures with stable interfaces.</a> BioTechniques. 55 (1), 21-26 (2013).</li><li>Tarone, G., Galetto, G., Prat, M., Comoglio, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+molecules+and+fibronectin-mediated+cell+adhesion:+effect+of+proteolytic+digestion+of+membrane+proteins.">Cell surface molecules and fibronectin-mediated cell adhesion: effect of proteolytic digestion of membrane proteins.</a> The Journal of cell biology. 94 (1), 179-186 (1982).</li><li>Trujillo, V., Kim, J., Hayward, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creasing+instability+of+surface-attached+hydrogels.">Creasing instability of surface-attached hydrogels.</a> Soft Matter. 4 (3), 564-569 (2008).</li><li>Saha, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+creasing+instability+of+soft+polyacrylamide+cell+culture+substrates.">Surface creasing instability of soft polyacrylamide cell culture substrates.</a> Biophysical journal. 99 (12), L94-L96 (2010).</li><li>Buxboim, A., Rajagopal, K., Andre&#8217;EX, B., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+deeply+cells+feel:+methods+for+thin+gels.">How deeply cells feel: methods for thin gels.</a> Journal of Physics: Condensed Matter. 22 (19), 194116 (2010).</li><li>Merkel, R., Kirchgessner, N., Cesa, C. M., Hoffmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+force+microscopy+on+elastic+layers+of+finite+thickness.">Cell force microscopy on elastic layers of finite thickness.</a> Biophysical journal. 93 (9), 3314-3323 (2007).</li></ol>

The authors have nothing to disclose.

Polyacrylamide gels, originally developed for electrophoresis,28 are now routinely used as cell culture substrates to study the effects of substrate stiffness on cell morphology, motility, and communication3,24,29 among other cell characteristics. Polyacrylamide allows manipulation of substrate stiffness to encompass the stiffness of all soft tissues in the body (0.3 &#8211; 300 kPa)1 with a simple change in polymer precursor concentration (Figure 2, Table 1, also see ref...

Most tissues in the body are soft viscoelastic materials with a Young&#8217;s modulus ranging from 0.1 kPa for brain to 100 kPa for soft cartilage, yet, most in vitro cell research is conducted on tissue culture polystyrene (TCP) which has a modulus of ~1 GPa.1 This stiffness mismatch greatly affects the way cells respond to their environment. A growing body of research is thus dedicated to elucidating the effect of substrate stiffness on the fate of various cell types,2,3 including stem cells.4 As a result, multiple hydrogels have been developed to aid in the understanding of stiffness-dependent cell biology including polyacrylamide (PA),5-7 polyethylene glycol (PEG),8,9 polydimethylsiloxane (PDMS),10 and alginate.11 While the evidence that substrate stiffness has a substantial impact on cell fate is growing, most studies are conducted on a small scale with a small number of samples. Systematic, multidimensional studies on the effect of substrate stiffness for an array of cell types or environmental conditions are rare.12
Several promising high-throughput hydrogel technologies have been developed, including PEG-based microarrays,13 microfluidic devices for the production of agarose hydrogel microbeads,14 or micro and nano-rods where stiffness is modulated by the diameter and height of the microrods.15 However, the technologies to prepare such substrates are sophisticated and available to limited number of laboratories. Much research involving stiffness modulated cell responses utilizes polyacrylamide (PA) gels which are not only inexpensive and simple to implement, but also exhibit a physiologically relevant range of Young&#8217;s modulus, namely 0.3 &#8211; 300 kPa.16-22 However, existing methods to fabricate PA gels for cell culture are labor intensive and consequently prepared in small batches. Some of the difficulties associated with the preparation of PA gels as cell substrates stem from the requirement that the gels have to be prepared: 1) in the absence of oxygen to allow complete polymerization, 2) with a flat and smooth surface to permit uniform cell attachment and spreading, and 3) permanently affixed to the bottom of the cell culture dish to prevent floating.
Several groups have attempted to produce PA gels for cell culture in large batches. Semler et al. prepared thick sheets of PA gels which were then &#8220;cut&#8221; with a hole punch and placed into 96-well plates.23 However, this method is limited to stiffer gels, i.e., &#62; 1 kPa in Young&#8217;s modulus, because softer gels are &#8220;sticky&#8221;, difficult to cut, and easily damaged. Mih et al. developed a more sophisticated technique which allows the gels to be directly polymerized in a glass-bottom multiwell plate.6 This was achieved by pouring the gel solutions into functionalized glass-bottom plates and forming gels by &#8220;sandwiching&#8221; them with a custom coverglass array.6 Even though very promising, slight edge effects were still observed with this technique. Additionally, the technique requires a custom-designed array not immediately accessible to many laboratories as well as costly glass-bottom multiwell plates.
This paper describes a simple and inexpensive way to assemble PA gels in a multiwell plate that could be easily adopted by any laboratory. Here, a flexible plastic support is utilized, which has two sides &#8212; a hydrophobic one, which is repellent to PA gels, and a hydrophilic one, which covalently binds the PA gel upon deposition. Once PA gel sheets are deposited and permanently affixed to the flexible plastic support, it enables handling gels of any thickness or stiffness and cutting them into any desired shape. This approach not only produces custom plastic &#8216;coverslips&#8217; in sizes not otherwise commercially available, but also obviates the necessity to pre-treat glass surfaces, either glass coverslips or the wells of costly glass-bottom multiwell plates, with a PA binding solution, which is a tedious and a time-consuming step. Lastly, uniform PA gels sheets can be prepared in large batches and stored de-hydrated for several months.
In summary, the assay presented here is an improvement over existing methods in several aspects. First, the process of multiwell plate assembly is efficient, and the overall cost of the required materials is low. Second, the hydrogels are produced in large batches in a single homogeneous gel film. Finally, only materials that are commercially available are required. The utility of the assay is illustrated by exploring the effect of substrate stiffness on cell morphology and spreading area.

<table border="0" cellpadding="0" cellspacing="0" width="898">
	<tbody>
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			<td height="21" style="height:21px;width:303px;">Reagents</td>
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		<tr height="20">
			<td height="20" style="height:21px;width:303px;">40% Acrylamide</td>
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			<td style="width:187px;">22589</td>
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			<td style="width:187px;">354236</td>
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			<td height="21" style="height:21px;width:303px;">Dimethyl sulfoxide (DMSO)</td>
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			<td style="width:187px;">BP231-100</td>
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		<tr height="20">
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			<td style="width:187px;">SH30256.01</td>
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			<td height="41" style="height:42px;width:303px;">Polydimehylsiloxane (PDMS) [Slygard 182 Elastomer Kit]</td>
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			<td style="width:187px;">3097358-1004</td>
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			<td height="21" style="height:21px;width:303px;">Tyrpsin/EDTA (10x)</td>
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			<td style="width:187px;">44174</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:303px;">RPMI-1640 Medium (1x)</td>
			<td style="width:200px;">HyClone</td>
			<td style="width:187px;">SH30027-02</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Fetal Bovine Serum</td>
			<td style="width:200px;">HyClone</td>
			<td style="width:187px;">SH30073-03</td>
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			<td height="21" style="height:21px;width:303px;">Penicillin Streptomycin</td>
			<td style="width:200px;">MP Biomedicals</td>
			<td style="width:187px;">1670046</td>
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			<td height="21" style="height:21px;width:303px;">Detergent - Triton-X</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">T8787</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Formaldehyde 37% Solution</td>
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			<td style="width:187px;">F1635</td>
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			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">A2153</td>
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			<td height="42" style="height:42px;width:303px;">BSA-based cell adhesion blocking kit - ECM Cell Adhesion Array Kit</td>
			<td style="width:200px;">Chemicon International</td>
			<td style="width:187px;">ECM540</td>
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			<td height="21" style="height:21px;width:303px;">Disposable lab equipment</td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
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			<td height="42" style="height:42px;width:303px;">flexible plastic support - GelBond PAG Film for Polyacrylamide Gels</td>
			<td style="width:200px;">GE Healthcare Bio-Sciences</td>
			<td style="width:187px;">309819</td>
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			<td height="21" style="height:21px;width:303px;">Glass Plates</td>
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			<td height="21" style="height:21px;width:303px;">50 mL Conicals</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">3181345107</td>
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			<td height="21" style="height:21px;width:303px;">15 mL Conicals</td>
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			<td height="21" style="height:21px;width:303px;">Micro centrifuge tubes</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">2 mL: 02681258</td>
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			<td height="21" style="height:21px;width:303px;">96-well plate (flat bottom)</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">12565501</td>
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			<td height="105" style="height:105px;width:303px;">Disposable Pipettes (1 mL, 2mL, 5mL, 10mL, 25 mL, 50mL)</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">1 mL: 13-678-11B, 2mL: 05214038, 5mL(FALCON): 357529, 10mL: 13-678-11E, 25mL: 13-678-11, 50mL: 13-678-11F</td>
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			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">5 3/4&#34;: 1367820A, 9&#34;:136786B</td>
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		<tr height="42">
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			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:187px;">13-711-9D</td>
			<td></td>
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			<td height="21" style="height:21px;width:303px;">Parafilm</td>
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			<td style="width:187px;">PM992</td>
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		<tr height="21">
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			<td style="width:200px;">Quest</td>
			<td style="width:187px;">92897</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Silicone spacers - Silicone sheet, 0.5 mm thick/13 cm x 18 cm</td>
			<td style="width:200px;">Grace Bio-Labs</td>
			<td style="width:187px;">JTR-S-0.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;"></td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
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			<td style="width:187px;"></td>
			<td></td>
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			<td height="42" style="height:42px;width:303px;">Light and Flourescent Microscope (Axiovert 200M)</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:187px;">3820005619</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Microscope Software</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:187px;">AxioVision Rel. 4.8.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">UV oven</td>
			<td style="width:200px;">UVITRON</td>
			<td style="width:187px;">UV1080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Vacuum chamber/degasser</td>
			<td style="width:200px;">BelArt</td>
			<td style="width:187px;">999320237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Vacuum pump for degasser</td>
			<td style="width:200px;">KNF Lab</td>
			<td style="width:187px;">5097482</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Tissue Culture Hood</td>
			<td style="width:200px;">NUAIRE</td>
			<td style="width:187px;">NU-425-600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Chemical Fume Hood</td>
			<td style="width:200px;">KEWAUNEE</td>
			<td style="width:187px;">99151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Inverted Microscope (Axiovert 25)</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:187px;">663526</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Incubator</td>
			<td style="width:200px;">NUAIRE</td>
			<td style="width:187px;">NU-8500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Pipette Aid</td>
			<td style="width:200px;">Drummond Scientific Co.</td>
			<td style="width:187px;">P-76864</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Hemacytometer</td>
			<td style="width:200px;">Bright-Line</td>
			<td style="width:187px;">383684</td>
			<td></td>
		</tr>
	</tbody>
</table>

simple polyacrylamide-based multiwell stiffness assay for the study of stiffness-dependent cell responses

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a matrix that is elastic and much softer (0.1 &#8211; 100 kPa). Since such stiffness mismatch greatly affects cell responses, there is a strong interest in developing hydrogel materials that span a wide range of stiffness to serve as cell substrates. Polyacrylamide gels, which are inexpensive and cover the stiffness range of all soft tissues in the body, are the hydrogel of choice for many research groups. However, polyacrylamide gel preparation is lengthy, tedious, and only suitable for small batches. Here, we describe an assay which by utilizing a permanent flexible plastic film as a structural support for the gels, enables the preparation of polyacrylamide gels in a multiwell plate format. The technique is faster, more efficient, and less costly than current methods and permits the preparation of gels of custom sizes not otherwise available. As it doesn&#8217;t require any specialized equipment, the method could be easily adopted by any research laboratory and would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Polyacrylamide (PA) hydrogels are widely used to test stiffness-dependent cell responses.17,24 By mixing various concentrations of acrylamide (A) and bis-acrylamide (B) one can make PA gels that span the stiffness range of most soft tissues in the body &#8212; 0.3 &#8211; 300 kPa Young&#8217;s modulus.1 However, preparation of polyacrylamide gels is tedious and time consuming, often limiting their usefulness in &#8220;high-throughput&#8221; applications such as for example drug screening.12</su...

Watch this Scientific Journal Video about Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses at JoVE.com

Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a ...

simple-polyacrylamide-based-multiwell-stiffness-assay-for-study

Research

JoVE Journal

Bioengineering

19.5K 次观看.  Saint Louis University. Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

视频: Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates. It is ideal for medium- to high-throughput screens in academic settings. It has an integrated fluid transfer module equipped with a multi-channel pipetter and the machine reads one column at a time to monitor fluorescence changes of a variety of fluorescent reagents. For example, FlexStation 3 has been used to study the function of Ca2+-permeable ion channels and G-protein coupled receptors by measuring the changes of intracellular free Ca2+ levels. Transient receptor potential (TRP) channels are a large family of nonselective cation channels that play important roles in many physiological and pathophysiological functions. Most of the TRP channels are calcium permeable and induce calcium influx upon activation. In this video, we demonstrate the application of FlexStation 3 to study the pharmacological profile of the TRPA1 channel, a molecular sensor for numerous noxious stimuli. HEK293 cells transiently or stably expressing human TRPA1 channels, grown in 96-well plates, are loaded with a Ca2+-sensitive fluorescent dye, Fluo-4, and real-time fluorescence changes in these cells are measured before and during the application of a TRPA1 agonist using the FLEX mode of the FlexStation 3. The effect of a putative TRPA1 antagonist was also examined. Data are transferred from the SoftMax Pro software to construct concentration-response relationships of TRPA1 activators and inhibitors.

This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

A Simple and Scalable Fabrication Method for Organic Electronic Devices on Textiles

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices cannot be worn under everyday conditions. Therefore, textiles are commonly considered the best substrate to accommodate electronic devices in wearable use. In this paper, we describe how to selectively pattern organic electroactive materials on textiles from a solution in an easy and scalable manner. This versatile deposition technique enables the fabrication of wearable organic electronic devices on clothes.

In this paper, we present a protocol to selectively deposit organic materials on textiles, which allows for the direct integration of organic electronic devices with wearables. The fabricated devices can be fully integrated in textiles, respecting their mechanical appearance and enabling sensing capabilities.

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...