This work was supported by grants from the National Institutes of Health (ES07408 and EB00208).

1. ECIS/Taxis Electrode Preparation
<ol>
 <li>The gold surface of the ECIS/taxis electrode array (consisting of 8 chambers per slide) is first stabilized by pre-treatment with sterile 10 mM cysteine in deionized water (dH2O) for 15 min at room temperature under sterile conditions.</li>
 <li>Aspirate the cysteine solution from each electrode chamber, rinse 3 times with sterile dH2O, and replace with 250 &mu;l of complete medium (RPMI 1640, 10%FBS, 25 mM HEPES buffer).</li>
 <li>Connect the electrode array to contact pins on instrument array holder to perform electrode check.</li>
 <li>Chambers that are properly connected will be highlighted in green on the computer screen. If the chambers are highlighted in red, they are not connected and the electrode should be re-positioned in the array holder to establish contacts for all chambers.</li>
 <li>Once all chambers are connected, click on the "check" button in the software controller to determine the initial resistance and capacitance values for each chamber. Resistance values should be between 1600 and 2000 ohms, and capacitance values should be between 5 and 6 nanofarads for an 8 chamber array. If a chamber is not within these acceptable parameters, the individual chamber should not be used to collect data.</li>
 <li>Disconnect the electrode from array holder to prepare the individual agarose chambers. Aspirate medium from chambers and wash each chamber 3 times with sterile dH2O.</li>
</ol>
2. Preparing Agarose Chambers
<ol>
 <li>In a sterile 15 ml polypropylene conical tube, measure 0.03 g of Seakem GTG agarose and add 1 ml sterile PBS. Replace tube cap loosely, and melt the agarose in autoclave for a 2 min liquid cycle (melting in a microwave is not effective for this assay). This short time will not melt the conical tube. For less fastidious cells that do not require serum for culture, (e.g. Dictyostelium discoideum) 0.5% agarose can be made in the appropriate medium and melted in a microwave. The autoclave is necessary for cells that require serum, as this will be denatured if melted in microwave.</li>
 <li>Add 5 ml of complete RPMI medium warmed in a water bath (55 &deg;-60 &deg;C) directly to the hot agarose, to a final concentration of 0.5% agarose and pipet up and down gently to mix.</li>
 <li>Pipet 300 &mu;l of melted agarose medium into each chamber, replace the ECIS/taxis array cover and allow to gel at room temperature.</li>
 <li>Pipet remaining agarose into cap of 15 ml tube to assess agarose gelling. This excess agarose will also be used in part 4: Cutting wells into agarose.</li>
</ol>
3. Constructing and Sharpening the Cannula Well Cutting Tool
<ol>
 <li>Sharpen two 14 gauge blunt end cannulae, using a 5/64" bit. Rotate the tip of the drill bit in each cannula opening with a slow speed drill, making certain the drill bit cuts entire interior edge of each cannula opening to form a sharp cutting edge (Figure 4A).</li>
 <li>Drill two holes through 1"x 2"x &frac14;" plexiglass (Figure 4B-D) with a 5/64" bit in a drill press. Insert each sharpened 14 gauge cannula so it protrudes an equal distance through each plexiglass hole.</li>
</ol>
4. Cutting Wells into Agarose
<ol>
 <li>Sterilize the 14 gauge cannulae by lightly flaming the tips. Prior to cutting wells in the ECIS chamber, allow the cannulae to cool to room temperature, or cool the cannulae by touching the agarose in the cap of the 15 ml tube. If the temperature of cannula is too high, the agarose will melt as the chamber wells are cut, rendering the wells misshapen and defective.</li>
 <li>Align the cannula pair above the two gold dots surrounding the target electrode in each chamber (Figure 2c and 5). Insert the cannula vertically, without any horizontal movement, stopping upon gentle contact between the cannula and the electrode surface. Carefully remove the cannula, again without any horizontal movement. Placement of additional wells for opposing gradients is also possible with a reconfiguration of the cannula jig.</li>
 <li>Gently aspirate the agarose plug left by the cannula, using a sterile 9" Pasteur pipette, connected to low vacuum pressure, with a vacuum trap catch agarose waste.</li>
 <li>Add 300 &mu;l of complete RPMI media to the surface of each chamber at 37&deg;C, for 2 min to saturate the agarose, and then gently aspirate the media from the surface and the wells without disturbing the agarose.</li>
</ol>
5. Loading the Wells
<ol>
 <li>Each well is capable of holding a volume of 7 &mu;l. To one well in each chamber, add 7 &mu;l of cells suspension. For Jurkat T cells, use 2 x107 cells/ml. These cells will respond to 7 &mu;l of 200 ng/ml SDF-1&alpha; diluted in incomplete RPMI, which lacks serum. Use complete media alone as a negative control.</li>
 <li>After the wells are loaded, view each chamber of the array with an inverted microscope to ensure that all wells were cut and filled properly. Cells should be in within the confines of the well's boundary. If they are seen in both wells, or are spreading beyond cell well border, then a gap has formed under the agarose. Note the improperly set up wells, as data cannot be reliably interpreted from those chambers.</li>
</ol>
6. Data Collection
<ol>
 <li>Place the electrode in the array holder and re-connect the gold pads on the electrode array to the spring-loaded pogo pins of the holder and click "setup". The wells that are properly connected will be highlighted in green. If the wells are red, they are not connected and the electrode should be repositioned in the array holder.</li>
 <li>Select "8WE1" (8 well 1 electrode) from the drop down menu in the software panel to indicate the array configuration.</li>
 <li>Select "check" in the software control panel to determine the initial resistance and capacitance values for each well. The resistance values should be between 1600 and 2000 ohms, while the capacitance values should be between 5 and 6 nanofarads.</li>
 <li>Select multiple frequencies to measure resistance at several frequencies. There is a choice of customizing frequencies, or using a set of recommended frequencies. The standard multiple frequency acquisition settings capture data at 11 pre-set frequencies (52.5, 125, 250, 500, 1,000, 2,000, 4,000, 8,000, 16,000, 32,000 and 64,000 Hz) at a rate of 8 wells/min. The time interval between measurements can also be customized. Collecting multiple frequency data allows for analysis of capacitance, impedance, and resistance. Collecting resistance values at 4,000 Hz is usually sufficient for mammalian cell chemotaxis studies, but the additional frequencies may provide information in other contexts12,13.</li>
 <li>Total experiment duration can be set, on the right hand panel, or the experiment can be stopped manually, by clicking "finish" once cells have produced resistance that are indicative of arrival at the electrode. The time it takes for cells to arrive at the electrode depends on the cell type used.</li>
 <li>Click "Start" to initiate data collection.</li>
</ol>
7. Data Management
<ol>
 <li>Although the data can be exported to Excel, the ECIS 1600R software, which accompanies the ECIS&theta; system is the most efficient method to analyze ECIS/Taxis data.</li>
 <li>The software data analysis tool bar contains the radio buttons Z, R, and C. These determine whether impedance, resistance, or capacitance, respectively, is plotted on the vertical axis against the x-axis (elapsed time).</li>
 <li>ECIS/Taxis data is most efficiently displayed as resistance over time at 4,000 Hz. Rapid fluctuations in resistance, or microtransients (Figure 1), are evidence of cellular movement over the electrode. Compiling, or binning the data points so that not all points are graphed, is not recommended for ECIS/Taxis, since it will average microtransients out of the data.</li>
 <li>Once the graph has been made with the ECIS1600R software, it can be exported by choosing "export graph" from the file menu, and saved as a .tif or .jpg file.</li>
</ol>
8. Representative Results
Cellular chemotaxis is indicated by an increase in resistance at 4,000 Hz. The arrival of cells on the target electrode is also indicated by the appearance of rapid fluctuations in resistance called microtransients, or micromotion as described in Opp et. al., 2009 (Figure 1)14. A negative result is indicated by consistent, or a slight decrease, in resistance at 4,000 Hz, as seen in red in Figure 1. The absence of microtransients is also a sign of an absence of cell movement to the electrode. The increase in resistance is directly proportional to the number cells that cross the electrode10. The addition of monoclonal antibody specific for a chemoattractant or toxins known to interfere with G protein coupled receptors can block chemotaxis11.
 
<img alt="Figure 1" src="/files/ftp_upload/3840/3840fig1.jpg" /> 
Figure 1. Chemotaxis of Human Jurkat T cells in response to SDF1&alpha;. Human Jurkat T cells (2.0x106 cell/ml) were exposed to a gradient of SDF-1&alpha; (starting concentration 100 ng/ml) or RPMI 1640 as a negative control. The normalized resistance at 4000 Hz was graphed. Human Jurkat T cells moved in response to SDF-1&alpha;, as evidenced by the increase in resistance and microtransients, while no movement was seen with exposure to RPMI 1640.
<img alt="Figure 2" src="/files/ftp_upload/3840/3840fig2.jpg" /> 
Figure 2. Top view of an ECIS/Taxis Electrode without agarose. Each ECIS/Taxis electrode is comprised of 8 individual chambers. Each chamber shares a large electrode (a), and contains an individual target electrode (b). Each chamber also has 2 gold circles (c), which are used as a guide when cutting the wells in the agarose with the cannulae. The gold squares (d) are the contact pads that connect to the pogo pins in order to apply the AC current to the electrode. The individual target electrodes are each in circuit with larger counter electrode (e).
<img alt="Figure 3" src="/files/ftp_upload/3840/3840fig3.jpg" /> 
Figure 3. Two Wells are cut into each agarose-filled chamber. When the chemoattractant is added to one well it diffuses to create a gradient in the agarose, with the highest concentration of chemoattract closest to the chemoattractant well. The cells travel beneath the agarose toward higher concentrations of chemoattractant and can pass over the target electrode. As the cells cross the target electrode, an increase of resistance is recorded by the ECIS 1600R software.
<img alt="Figure 4" src="/files/ftp_upload/3840/3840fig4.jpg" /> 
Figure 4. Insertion of two 14 gauge cannulae into plexiglass. A) A 5/64" drill bit is used to sharpen the cannula tip. B) Two holes must be drilled into plexiglass to accommodate the 14 gauge cannulae according to the layout shot. C) Use a drill press to ensure the holes are perpendicular to the pleixglass surface. D) Two sharpened Cannulae are inserted through &frac14;" plexiglass, 2 mm apart (measured from inner edges). These sharpened cannulae are carefully inserted into agarose to cut the wells.
<img alt="Figure 5" src="/files/ftp_upload/3840/3840fig5.jpg" /> 
Figure 5. Aligning cannulae with gold dots in ECIS/Taxis chamber. To cut wells, the sharpened cannulae must be aligned with the gold dots flanking the target electrode on the bottom of the ECIS/Taxis chamber. The cannulae should be inserted vertically, without any horizontal movement, and removed in the same manner.

<ol>
	<li>Boyden, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemotactic+effect+of+mixtures+of+antibody+and+antigen+on+polymorphonuclear+leucocytes.">The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes.</a> J. Exp. Med. 115, 453-466 (1962).</li><li>Lauffenburger, D. A., Tranquillo, R. T., Zigmond, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+gradients+of+chemotactic+factors+in+chemotaxis+assays.">Concentration gradients of chemotactic factors in chemotaxis assays.</a> Methods Enzymol. 162, 85-101 (1988).</li><li>Nelson, R. D., Quie, P. G., Simmons, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+under+agarose:+a+new+and+simple+method+for+measuring+chemotaxis+and+spontaneous+migration+of+human+polymorphonuclear+leukocytes+and+monocytes.">Chemotaxis under agarose: a new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes.</a> J. Immunol. 115, 1650-1650 (1975).</li><li>Newton-Nash, D. K., Tonellato, P., Swiersz, M., Abramoff, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+chemokinetic+behavior+of+inflammatory+lung+macrophages+in+a+linear+under-agarose+assay.">Assessment of chemokinetic behavior of inflammatory lung macrophages in a linear under-agarose assay.</a> J. Leukoc. Biol. 48, 297-305 (1990).</li><li>Giaever, I., Keese, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+fibroblast+behavior+in+tissue+culture+with+an+applied+electric+field.">Monitoring fibroblast behavior in tissue culture with an applied electric field.</a> Proc. Natl. Acad. Sci. U.S.A. 81, 3761-374 (1984).</li><li>Keese, C. G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Whole+Cell+Biosensor+bsed+on+Cell-Substrate+Interactions.">A Whole Cell Biosensor bsed on Cell-Substrate Interactions.</a> IEEE Engineering in Medicine and Biology Society. 12, 500-501 (1990).</li><li>Laevsky, G., Knecht, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-linking+of+actin+filaments+by+myosin+II+is+a+major+contributor+to+cortical+integrity+and+cell+motility+in+restrictive+environments.">Cross-linking of actin filaments by myosin II is a major contributor to cortical integrity and cell motility in restrictive environments.</a> J. Cell. Sci. 116, 3761-3770 (2003).</li><li>Condeelis, J., Bresnick, A., Demma, M., Dharmawardhane, S., Eddy, R., Hall, A. L., Sauterer, R., Warren, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+amoeboid+chemotaxis:+an+evaluation+of+the+cortical+expansion.">Mechanisms of amoeboid chemotaxis: an evaluation of the cortical expansion.</a> 11, 5-6 (1990).</li><li>Hadjout, N., Laevsky, G., Knecht, D. A., Lynes, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+real-time+measurement+of+chemotactic+cell+motility.">Automated real-time measurement of chemotactic cell motility.</a> Biotechniques. 31, 1130-1138 (2001).</li><li>Hadjout, N., Yin, X., Knecht, D. A., Lynes, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+real-time+measurements+of+leukocyte+chemotaxis.">Automated real-time measurements of leukocyte chemotaxis.</a> J. Immunol. Methods. 320, 70-80 (2007).</li><li>Yin, X., Knecht, D. A., Lynes, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metallothionein+mediates+leukocyte+chemotaxis.">Metallothionein mediates leukocyte chemotaxis.</a> BMC Immunol. 6, 21-21 (2005).</li><li>Lundien, M. C., Mohammed, K. A., Nasreen, N., Tepper, R. S., Hardwick, J. A., Sanders, K. L., Van Horn, R. D., Antony, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+MCP-1+expression+in+airway+epithelial+cells:+role+of+CCR2+receptor+in+airway+epithelial+injury.">Induction of MCP-1 expression in airway epithelial cells: role of CCR2 receptor in airway epithelial injury.</a> J. Clin. Immunol. 22, 144-152 (2002).</li><li>Zudaire, E., Cuesta, N., Murty, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+aryl+hydrocarbon+receptor+repressor+is+a+putative+tumor+suppressor+gene+in+multiple+human+cancers.">The aryl hydrocarbon receptor repressor is a putative tumor suppressor gene in multiple human cancers.</a> J. Clin. Invest. 118, 640-650 (2008).</li><li>Opp, D., Wafula, B., Lim, J., Huang, E., Lo, J. C., Lo, 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=Use+of+electric+cell-substrate+impedance+sensing+to+assess+in+vitro+cytotoxicity.">Use of electric cell-substrate impedance sensing to assess in vitro cytotoxicity.</a> Biosens. Bioelectron. 24, 2625-269 (2009).</li><li>Foxman, E. F., Kunkel, E. J., Butcher, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+conflicting+chemotactic+signals.+The+role+of+memory+in+leukocyte+navigation.">Integrating conflicting chemotactic signals. The role of memory in leukocyte navigation.</a> J. Cell. Biol. 147, 577-588 (1999).</li><li>Heit, B., Tavener, S., Raharjo, E., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+intracellular+signaling+hierarchy+determines+direction+of+migration+in+opposing+chemotactic+gradients.">An intracellular signaling hierarchy determines direction of migration in opposing chemotactic gradients.</a> J. Cell. Biol. 159, 91-102 (1999).</li></ol>

David A Knecht and Michael A Lynes have an issued patent for the ECIS/Taxis technology, which as been licensed by the University of Connecticut to Applied Biophysics, Inc.

Novel characteristics of the ECIS/Taxis assay include its ability to automate the collection of real-time data as cells respond to chemoattractant. While the most commonplace application of this technology is to measure cellular responses to individual chemotactic gradients, or to gradients comprised of mixtures of chemotaxis agonists and antagonists, the ECIS/Taxis approach is also amenable to variations to these configurations that could be quite helpful in the assessment of cellular responsiveness. There is good evide...

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog number </td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>ECIS Z&theta;</td>
			<td>Applied Biophysics</td>
			<td>&nbsp;</td>
			<td><a href="http://www.biophysics.com/prodducts_Ecisz0.php">http://www.biophysics.com/prodducts_Ecisz0.php</a></td>
		</tr>
		<tr>
			<td>ECIS Electrode Array</td>
			<td>Applied Biophysics</td>
			<td>8W Chemotaxis</td>
			<td><a href="http://www.biophysics.com/cultureware.php">http://www.biophysics.com/cultureware.php</a></td>
		</tr>
		<tr>
			<td>Seakem GTG agarose</td>
			<td>BioWhittaker Molecular Applications</td>
			<td>50070</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RPMI1640</td>
			<td>Cellgro</td>
			<td>10-040</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HyClone Fetal Bovine Serum</td>
			<td>Thermo Scientific</td>
			<td>SH300703</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>MP Biomedicals</td>
			<td>1670049</td>
			<td>Penicillin 5,000 IU/ml; Streptomycin 5 mg/ml</td>
		</tr>
		<tr>
			<td>HEPES Buffer</td>
			<td>MP Biomedicals</td>
			<td>1688449</td>
			<td>1M solution, cell culture grade</td>
		</tr>
		<tr>
			<td>14 Gauge stainless steel Cannula (2) 4 inch</td>
			<td>General Laboratory Supply</td>
			<td>5-8365-1</td>
			<td>Blunt point</td>
		</tr>
	</tbody>
</table>

measurement of cellular chemotaxis with ecis/taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

Watch this Scientific Journal Video about Measurement of Cellular Chemotaxis with ECIS/Taxis at JoVE.com

Measurement of Cellular Chemotaxis with ECIS/Taxis

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

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Biology

14.7K 次观看.  University of Connecticut. 企业家信心指数/出租车系统是一个自动化的，实时的检测，测量细胞趋化。在这个实验中，细胞的琼脂糖层到达目标电极下方移动。细胞运动耐交流电流0开始测量。

视频: Measurement of Cellular Chemotaxis with ECIS/Taxis

Studies of Bacterial Chemotaxis Using Microfluidics - Interview

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades. Today, such pipettes are made from glass capillaries using a programmable puller. Such instruments heat the capillary using either a metal filament or a laser and draw out the glass using gravity, a motor or both. Pipettes for patch-clamp recording are formed using only heat and gravity, while sharp electrodes for intracellular recording use a combination of heat, gravity, and a motor. The procedure used to make intracellular recording pipettes is similar to that used to make injection needles for a variety of applications, including cRNA injection into Xenopus oocytes. In general, capillary glass &lt;1.2 mm in diameter is used to make pipettes for patch clamp recording, while narrower glass is used for intracellular recording (outer diameter = 1.0 mm). For each tool, the puller is programmed slightly differently. This video shows how to make both kinds of recording pipettes using pre-established puller programs.

This video shows how to use a programmable puller to make patch pipettes and sharp electrodes for electrophysiology. The same procedure can be used to make a variety of glass tools, including injection needles.

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades.  Today, such pipettes are made from glass capillaries ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG

Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity [1]. The electrophysiological activity of the brain generates electric fields  - that can be recorded using electroencephalography (EEG)- and their concomitant magnetic fields  - detected by MEG. MEG signals are detected by specialized sensors known as superconducting quantum interference devices (SQUIDs). Superconducting sensors require cooling with liquid helium at -270 &deg;C. They are contained inside a vacumm-insulated helmet called a dewar, which is filled with liquid. SQUIDS are placed in fixed positions inside the helmet dewar in the helium coolant, and a subject's head is placed inside the helmet dewar for MEG measurements. The helmet dewar must be sized to satisfy opposing constraints. Clearly, it must be large enough to fit most or all of the heads in the population that will be studied. However, the helmet must also be small enough to keep most of the SQUID sensors within range of the tiny cerebral fields that they are to measure. Conventional whole-head MEG systems are designed to accommodate more than 90% of adult heads. However adult systems are not well suited for measuring brain function in pre-school chidren whose heads have a radius several cm smaller than adults. The KIT-Macquarie Brain Research Laboratory at Macquarie University uses a MEG system custom sized to fit the heads of pre-school children. This child system has 64 first-order axial gradiometers with a 50 mm baseline[2] and is contained inside a magnetically-shielded room (MSR) together with a conventional adult-sized MEG system [3,4]. There are three main advantages of the customized helmet dewar for studying children. First, the smaller radius of the sensor configuration brings the SQUID sensors into range of the neuromagnetic signals of children's heads. Second, the smaller helmet allows full insertion of a child's head into the dewar. Full insertion is prevented in adult dewar helmets because of the smaller crown to shoulder distance in children. These two factors are fundamental in recording brain activity using MEG because neuromagnetic signals attenuate rapidly with distance. Third, the customized child helmet aids in the symmetric positioning of the head and limits the freedom of movement of the child's head within the dewar. When used with a protocol that aligns the requirements of data collection with the motivational and behavioral capacities of children, these features significantly facilitate setup, positioning, and measurement of MEG signals.

The advent of MEG systems sized for young children opens important new opportunities to study brain development. The new system, together with a protocol that aligns experimental requirements with the capacities of children, can be used to study cognitive and language processes in healthy, awake children aged three to six.

Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity [1]. The electrophysiological activity of the brain ...

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...