This work is supported by NIH R01 GM47607, CA163296 and NSF 1334493 to CET. We thank members of the Turner lab for critical reading of the manuscript. All data shown in this report were reproduced by permission from Wormer et al. 201411.

1. Preparation of Durotaxis Chambers
<ol>
	<li>To prepare one 6-well tissue culture plate, tare the balance with a 50-ml conical tube. Weigh out approximately 10 g of the PDMS base solution in the 50-ml tube (the solution is quite viscous).</li>
	<li>For a 90:1 substrate (Compliance of ~1 kPa), divide the measured weight of the PDMS base solution in the tube by 90 to determine the correct amount of crosslinker solution needed. Add the calculated amount of the PDMS crosslinker solution to the same tube. 
	Example: 10 g / 90 = 0.11 g. Add 0.11 g of the PDMS crosslinker solution to the PDMS base solution. 
	NOTE: The base and crosslinker solutions are both provided in the PDMS kit.</li>
	<li>Vigorously mix the PDMS base/crosslinker mixture for 5 min at RT using a small spatula. At this stage the mixture will contain a large number of air bubbles.</li>
	<li>Centrifuge the PDMS substrate in a benchtop centrifuge for 5 min at 50 x g at RT to remove the bubbles. If there are still bubbles after the 5 min, centrifuge again.</li>
	<li>Pipette 1 ml of 90:1 PDMS substrate into each well of the tissue culture treated 6-well plate. Any remaining air bubbles present in the PDMS can be eliminated at this stage by popping them using a 21 G&#160;needle. Allow the PDMS to spread for 30 min in the well.</li>
	<li>Boil 12 mm glass #1 coverslips in distilled water for 5 min. Repeat two times and store the coverslips in distilled water.</li>
	<li>Place one, dried coverslip into each well of the tissue culture plate by gently touching one side of the coverslip into the PDMS solution then drop the coverslip onto the PDMS. As the coverslip settles, the PDMS will begin to encroach over the edges of the coverslip, but will not completely cover it. This will generate an interface between the PDMS and glass after curing (see Figure 1A,B).</li>
	<li>Incubate the plate at 70 &#176;C in an oven for 16 hr to cure (harden) the PDMS. Place the plate in a cell culture hood and UV sterilize for 10 min. 
	NOTE: It is best to make the plates within a couple days of use. However, the plates may be wrapped in Parafilm without any buffer and stored at 4 &#176;C for up to 2 weeks without any noticeable decrease in quality.</li>
</ol>
2. Cell Plating
NOTE: If studying the effect of siRNA-mediated knockdown, perform the knockdown using the manufacturer&#8217;s instructions or the optimized protocol for the cell type of choice.
<ol>
	<li>Coat each durotaxis chamber with 1 ml of 10 &#181;g/ml fibronectin in PBS without calcium and magnesium for 1 hr at 37 &#176;C. Alternatively, the fibronectin may be applied the day prior to analysis by incubating the PDMS with 10 &#181;g/ml fibronectin in PBS for 16 hr at 4 &#176;C. Make sure the entire surface is submerged in the fibronectin in PBS solution.</li>
	<li>Prepare heat-denatured 1% BSA. Weigh 0.5 g BSA and dissolve it in 50 ml of PBS. Filter sterilize the solution through a 0.22 &#181;m filter and heat at 80 &#176;C for 12 min. 
	Note: Prepare this solution the day prior to cell plating and store at 4 &#176;C.</li>
	<li>Aspirate the fibronectin solution and wash 3 times with PBS. Add 1 ml of heat-denatured BSA in PBS to each well and incubate for 30 min at RT.</li>
	<li>Trypsinize and count cells of choice while the durotaxis chambers are blocking with the heat denatured BSA.</li>
	<li>Plate 1 x 105 cells in a volume of 2 ml into each well of the durotaxis chamber, using the media required for the particular cell type of choice. Allow the cells to adhere and spread on the substrate for 4 hr in a humidified incubator at 37 &#176;C with 5% CO2. 
	NOTE: U2OS cells are routinely maintained in DMEM with 10% FBS, supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, and 10 I.U./ml penicillin, 10 &#181;g/ml streptomycin. 
	NOTE: The cell number used in this example is optimized for U2OS cells. Optimization for other cell types may be required. This density gives the cells enough room to migrate without significant interactions with other cells.</li>
</ol>
3. Live-cell Imaging
<ol>
	<li>Perform live cell imaging on an inverted microscope, using phase contrast with a 10X objective. The microscope should be fitted with an enclosed environmental, humidified chamber, allowing control of temperature at 37 &#176;C and 5% CO2 during long-term imaging.</li>
	<li>After the cells have spread for approximately 3.5 hr, assemble the plate in the microscope chamber. Allow the sample(s) to equilibrate in the chamber for 30 min.</li>
	<li>Set up automated, multi-point visiting on the microscope if available. Focus on the interface between the 90:1 PDMS and the glass coverslip and choose points to image all around the interface with an average of 40 points per durotaxis chamber. Image the cells every 10 min for up to 16 hr. 
	NOTE: The region of interest will appear as two lines, with the outer line corresponding to the edge of the coverslip and the inner line corresponding to the actual interface between the PDMS and the coverslip. See Figure 1B.</li>
</ol>
4. Data Analysis
<ol>
	<li>Generate a spreadsheet as in Table 1.</li>
	<li>Count the number of crossing events from the PDMS to the glass surface and vice versa from each movie generated. Record the number of crossing events in the appropriate column in the excel spreadsheet. 
	NOTE: A crossing event is defined as the cell nucleus passing over the boundary between the PDMS and glass in either direction.</li>
	<li>To quantify multiple crossings, count the number of times the cell crossed the interface. That number should be recorded in the excel column corresponding to the substrate on which the cell was located at the end of the movie. Repeat the analysis for every cell that crosses the interface in the movie. Exclude cells that migrate out of the field of view during imaging. 
	NOTE: It is also important to verify, by cell counting at the beginning of the experiment, that the cells are able to adhere equally to the fibronectin-coated PDMS and glass coverslip surfaces.</li>
	<li>Calculate the percentage of cells that migrated from PDMS to the glass surface (i.e., underwent durotaxis). Add the number of crossing events from soft to hard and the multiple crossing events that ended on hard and divide by the total number of crossing events.</li>
	<li>Calculate the percentage of multiple crossings by dividing the number of multiple crossings by the total number of crossings.</li>
</ol>

<ol>
	<li>Engler, A. J., Sen, S., Sweeney, H. L., 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=Matrix+elasticity+directs+stem+cell+lineage+specification.">Matrix elasticity directs stem cell lineage specification.</a> Cell. 126 (4), 677-689 (2006).</li><li>Paszek, M. J., 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=Tensional+homeostasis+and+the+malignant+phenotype.">Tensional homeostasis and the malignant phenotype.</a> Cancer Cell. 8 (3), 241-254 (2005).</li><li>Levental, K. R., 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=Matrix+crosslinking+forces+tumor+progression+by+enhancing+integrin+signaling.">Matrix crosslinking forces tumor progression by enhancing integrin signaling.</a> Cell. 139 (5), 891-906 (2009).</li><li>Lo, C. M., Wang, H. B., Dembo, M., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+movement+is+guided+by+the+rigidity+of+the+substrate.">Cell movement is guided by the rigidity of the substrate.</a> Biophysical Journal. 79 (1), 144-152 (2000).</li><li>Plotnikov, S. V., Waterman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guiding+cell+migration+by+tugging.">Guiding cell migration by tugging.</a> Current Opinion in Cell Biology. 25 (5), 619-626 (2013).</li><li>Choquet, D., Felsenfeld, D. P., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+rigidity+causes+strengthening+of+integrin-cytoskeleton+linkages.">Extracellular matrix rigidity causes strengthening of integrin-cytoskeleton linkages.</a> Cell. 88 (1), 39-48 (1997).</li><li>Balaban, N. Q., 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=Force+and+focal+adhesion+assembly:+a+close+relationship+studied+using+elastic+micropatterned+substrates.">Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates.</a> Nature Cell Biology. 3 (5), 466-472 (2001).</li><li>Provenzano, P. P., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+signaling+through+the+cytoskeleton+regulates+cell+proliferation+by+coordinated+focal+adhesion+and+Rho+GTPase+signaling.">Mechanical signaling through the cytoskeleton regulates cell proliferation by coordinated focal adhesion and Rho GTPase signaling.</a> Journal of Cell Science. 124 (8), 1195-1205 (2011).</li><li>Wang, Y. L., Pelham, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+a+flexible,+porous+polyacrylamide+substrate+for+mechanical+studies+of+cultured+cells.">Preparation of a flexible, porous polyacrylamide substrate for mechanical studies of cultured cells.</a> Methods in Enzymology. 298, 489-496 (1998).</li><li>Prager-Khoutorsky, M., 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=Fibroblast+polarization+is+a+matrix-rigidity-dependent+process+controlled+by+focal+adhesion+mechanosensing.">Fibroblast polarization is a matrix-rigidity-dependent process controlled by focal adhesion mechanosensing.</a> Nature Cell Biology. 13 (12), 1457-1465 (2011).</li><li>Wormer, D. B., Davis, K. A., Henderson, J. H., Turner, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+focal+adhesion-localized+CdGAP+regulates+matrix+rigidity+sensing+and+durotaxis.">The focal adhesion-localized CdGAP regulates matrix rigidity sensing and durotaxis.</a> PloS ONE. 9 (3), e91815 (2014).</li><li>Trichet, L., 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=Evidence+of+a+large-scale+mechanosensing+mechanism+for+cellular+adaptation+to+substrate+stiffness.">Evidence of a large-scale mechanosensing mechanism for cellular adaptation to substrate stiffness.</a> Proceedings of the National Academy of Sciences. 109 (18), 6933-6938 (2012).</li><li>Eyckmans, J., Boudou, T., Yu, X., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hitchhiker&#8217;s+guide+to+mechanobiology.">A hitchhiker&#8217;s guide to mechanobiology.</a> Developmental Cell. 21 (1), 35-47 (2011).</li><li>Martinac, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensitive+ion+channels:+molecules+of+mechanotransduction.">Mechanosensitive ion channels: molecules of mechanotransduction.</a> Journal of Cell Science. 117 (12), 2449-2460 (2004).</li><li>Jafar-Nejad, H., 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=Sec15,+a+component+of+the+exocyst,+promotes+notch+signaling+during+the+asymmetric+division+of+Drosophila+sensory+organ+precursors.">Sec15, a component of the exocyst, promotes notch signaling during the asymmetric division of Drosophila sensory organ precursors.</a> Developmental Cell. 9 (3), 351-363 (2005).</li></ol>

The authors have no conflicts to disclose.

Herein we describe a simple assay to study durotaxis in migrating cells. A major strength of this assay is the ease of preparing the durotaxis chambers using PDMS. The rigidity of the substrates can be easily manipulated by changing the ratio of PDMS base solution to crosslinker to allow the study of various rigidities in the assay. However, one potential limitation of the system is that cells are only exposed to a single change in substrate rigidity as opposed to experiencing a rigidity gradient that is provided by more...

The extracellular matrix (ECM) is comprised of a complex array of structural and crosslinking proteins including collagen, fibronectin and laminin. Although it is well established that the ECM provides important structural support for cellular tissues, there is increasing evidence to indicate that cells actively respond to physical changes in their ECM environment to regulate diverse cellular processes including cell survival, differentiation and cell migration. For example, differences in the rigidity of the ECM can drive mesenchymal stem cells towards different lineages, with soft substrates (~1 kPa) promoting neurogenic lineages while stiff (~25 kPa) substrates promote osteogenic differentiation1. Similarly, an increase in the stromal matrix rigidity has been shown to promote mammary epithelial cell tumorigenesis and invasion into the surrounding tissue2,3.
A particularly interesting aspect of this mechanosignaling activity results in a process known as durotaxis, in which cells migrate preferentially towards a more rigid substrate4,5. Cells constantly sense the physical characteristics of their extracellular environment through integrin receptor binding to the ECM. This, in turn, promotes the accumulation of numerous structural and signaling proteins to their cytoplasmic domains to drive the formation of adhesive structures known as focal adhesions or focal contacts6,7. Since integrins have no inherent enzymatic activity, signals are relayed from the ECM through these accessory proteins to coordinate the cell&#8217;s response to their changing environment8. Accordingly, the identification and characterization of the key proteins involved in regulating mechanosignaling and durotaxis is an important area of investigation.
Various model systems have been developed to study durotaxis in vitro, but most have utilized collagen-coated polyacrylamide substrates4. However, the preparation of the polyacrylamide substrates can be technically challenging and the collagen used in these assays must be chemically crosslinked to the substrate9. Polydimethylsiloxane (PDMS) substrates have been shown to exhibit comparable mechanical properties to the polyacrylamide substrates10. However, PDMS substrates are prepared by simply mixing a ratio of the base to crosslinker and these substrates can be coated with ECM proteins without the need for chemical crosslinking, thus making PDMS an easier tool to study the effects of rigidity on cell behavior. Herein, we describe how to prepare a simple durotaxis chamber in which a soft PDMS substrate is integrated with a rigid glass coverslip.
The assay, as outlined below, provides a quick and simple method to study durotaxis. For this study we used human U2OS osteosarcoma cells combined with siRNA-mediated knockdown of cdGAP to study the role of this focal adhesion protein in durotaxis11. Importantly, this protocol may be readily adapted to individual requirements. Other cell types may be substituted for the U2OS cells and any protein may be knocked down or overexpressed to determine the effects on cell behavior during durotaxis. Furthermore, this protocol may be adapted to incorporate fluorescently tagged proteins to analyze their dynamics and behavior using FRAP or FRET approaches.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning</td>
			<td>3097358-1004</td>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
		</tr>
		<tr>
			<td>#1 Cover glass 12mm</td>
			<td>Fisher Scientific</td>
			<td>12-545-82</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Celltreat</td>
			<td>229106</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Cellgro</td>
			<td>15-017-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Cellgro</td>
			<td>25-005-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Fisher Scientific</td>
			<td>BP356-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Cellgro</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>BD Biosciences</td>
			<td>610077</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>21600-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes</td>
			<td>Celltreat</td>
			<td>229456</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>U2OS cells</td>
			<td>ATCC</td>
			<td>HTB-96</td>
			<td></td>
		</tr>
	</tbody>
</table>

a simplified system for evaluating cell mechanosensing and durotaxis in vitro

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity greatly impacts cell behavior to regulate normal and pathologic processes including cell proliferation, differentiation, adhesion signaling and directional migration. In this regard, the innate ability of certain cell types to migrate towards a stiffer, or less compliant matrix substrate is referred to as durotaxis. This phenomenon plays an important role during embryonic development, wound repair and cancer cell invasion. Here, we describe a straightforward assay to study durotaxis, in vitro, using polydimethylsiloxane (PDMS) substrates. Preparation of the described durotaxis chambers creates a rigidity interface between the relatively soft PDMS gel and a rigid glass coverslip. In the example provided, we have used these durotaxis chambers to demonstrate a role for the cdc42/Rac1 GTPase activating protein, cdGAP, in mechanosensing and durotaxis regulation in human U2OS osteosarcoma cells. This assay is readily adaptable to other cell types and/or knockdown of other proteins of interests to explore their respective roles in mechanosignaling and durotaxis.

A schematic of the durotaxis chamber is shown in Figure 1A. Soft PDMS substrate (a 90:1 mixture of PDMS base to crosslinker solutions) is spread in a 6 well dish and a glass coverslip is placed on top of the PDMS, which then partially covers the upper surface of the coverslip, thereby creating an interface between the two substrates of different compliance. The rigidity of the soft PDMS substrate is ~1 kPa, which is comparable to the typical compliance of brain tissue, while the rigidity of glass is arou...

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A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...

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8.1K Views.  SUNY Upstate Medical University. Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

Video: A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non coding sequences. One prominent function carried out by the non coding fraction of the genome is to regulate gene transcription; however, there are no effective methods to broadly predict cis-regulatory elements from primary DNA sequence. We have developed an efficient protocol to functionally evaluate potential cis-regulatory elements through zebrafish transgenesis. Our approach offers significant advantages over cell-culture based techniques for developmentally important genes, since it provides information on spatial and temporal gene regulation. Conversely, it is faster and less expensive than similar experiments in transgenic mice, and we routinely apply it to sequences isolated from the human genome. Here we demonstrate our approach to selecting elements for testing based on sequence conservation and our protocol for cloning sequences and microinjecting them into zebrafish embryos.

We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish transgenesis.

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular &#8220;tags&#8221; for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate underlying a cell must be manipulated. While hydrogels with graded stiffness and long-term migration assays have proven useful in durotaxis studies, immediate, acute responses to local changes in substrate tension allow focused study of individual cell movements and subcellular signaling events. To repeatably test the ability of cells to sense and respond to the underlying substrate stiffness, a modified method for application of acute gradients of increased tension to individual cells cultured on deformable hydrogels is used which allows for real time manipulation of the strength and direction of stiffness gradients imparted upon cells in question. Additionally, by fine tuning the details and parameters of the assay, such as the shape and dimensions of the micropipette or the relative position, placement, and direction of the applied gradient, the assay can be optimized for the study of any mechanically sensitive cell type and system. These parameters can be altered to reliably change the applied stimulus and expand the functionality and versatility of the assay. This method allows examination of both long term durotactic movement as well as more immediate changes in cellular signaling and morphological dynamics in response to changing stiffness.

Mechanical forces are important for controlling cell migration. This protocol demonstrates the use of elastic hydrogels that can be deformed using a glass micropipette and a micromanipulator to stimulate cells with a local stiffness gradient to elicit changes in cell structure and migration.

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate ...

Assessment of Phytochemical Effects on &lt;em&gt;Helicoverpa armigera&lt;/em&gt; Through Feeding Assay

Developing a Feeding Assay System for Evaluating the Insecticidal Effect of Phytochemicals on Helicoverpa armigera

Helicoverpa armigera, a lepidopteran insect, is a polyphagous pest with a worldwide distribution. This herbivorous insect is a threat to plants and agricultural productivity. In response, plants produce several phytochemicals that negatively impact the insect's growth and survival. This protocol demonstrates an obligate feeding assay method to evaluate the effect of a phytochemical (quercetin) on insect growth, development, and survival. Under controlled conditions, the neonates were maintained until the second instar on a pre-defined artificial diet. These second-instar larvae were allowed to feed on a control and quercetin-containing artificial diet for 10 days. The insects' body weight, developmental stage, frass weight, and mortality were recorded on alternate days. The change in body weight, the difference in feeding pattern, and developmental phenotypes were evaluated throughout the assay time. The described obligatory feeding assay simulates a natural mode of ingestion and can be scaled up to a large number of insects. It permits one to analyze phytochemicals' effect on the growth dynamics, developmental transition, and overall fitness of H. armigera. Furthermore, this setup can also be utilized to evaluate alterations in nutritional parameters and digestive physiology processes. This article provides a detailed methodology for feeding assay systems, which may have applications in toxicological studies, insecticidal molecule screening, and understanding chemical effects in plant-insect interactions.

Author Spotlight: Development and Challenges of Feeding Assays for Insect Pest Management 

This protocol describes the obligate feeding assay to evaluate the potentially toxic effect of a phytochemical on the lepidopteran insect larvae. This is a highly scalable insect bioassay, easy to optimize the sublethal and lethal dose, deterrent activity, and physiological effect. This could be used for screening eco-friendly insecticides.

Helicoverpa armigera, a lepidopteran insect, is a polyphagous pest with a worldwide distribution. This herbivorous insect is a threat to plants and ...

Investigating Zinc Transport Mechanism in Mammalian Cells with FluoZin-3 Assay

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

Author Spotlight: Exploring Cellular Zinc Regulation Through ZnT1 Functionality 

Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...

Quantitative Analysis of Mitophagy in Murine Pancreatic &amp;#946;-Cells

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic &#946;-Cells

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient insulin release. Advanced quantitative assessments of mitophagy often require the use of genetic reporters. The mt-Keima mouse model, which expresses a mitochondria-targeted pH-sensitive dual-excitation ratiometric probe for quantifying mitophagy via flow cytometry, has been optimized in &#946;-cells. The ratio of acidic-to-neutral mt-Keima wavelength emissions can be used to robustly quantify mitophagy. However, using genetic mitophagy reporters can be challenging when working with complex genetic mouse models or difficult-to-transfect cells, such as primary human islets. This protocol describes a novel complementary dye-based method to quantify &#946;-cell mitophagy in primary islets using MtPhagy. MtPhagy is a pH-sensitive, cell-permeable dye that accumulates in the mitochondria and increases its fluorescence intensity when mitochondria are in low pH environments, such as lysosomes during mitophagy. By combining the MtPhagy dye with Fluozin-3-AM, a Zn2+ indicator that selects for &#946;-cells, and Tetramethylrhodamine, ethyl ester (TMRE) to assess mitochondrial membrane potential, mitophagy flux can be quantified specifically in &#946;-cells via flow cytometry. These two approaches are highly complementary, allowing for flexibility and precision in assessing mitochondrial quality control in numerous &#946;-cell models.

Author Spotlight: Assessment of Mitophagy Flux in Pancreatic &amp;#946;-Cells Using Effective and Robust Complementary Approaches 

This protocol outlines two methods for the quantitative analysis of mitophagy in pancreatic &#946;-cells: first, a combination of cell-permeable mitochondria-specific dyes, and second, a genetically encoded mitophagy reporter. These two techniques are complementary and can be deployed based on specific needs, allowing for flexibility and precision in quantitatively addressing mitochondrial quality control.

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient ...