1Molecular and Cell Biology, University of Connecticut, 2University of Connecticut
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Pietrosimone, K. M., Yin, X., Knecht, D. A., Lynes, M. A. Measurement of Cellular Chemotaxis with ECIS/Taxis. J. Vis. Exp. (62), e3840, doi:10.3791/3840 (2012).
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 SDF1α and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1α is shown in Figure 1.
1. ECIS/Taxis Electrode Preparation
2. Preparing Agarose Chambers
3. Constructing and Sharpening the Cannula Well Cutting Tool
4. Cutting Wells into Agarose
5. Loading the Wells
6. Data Collection
7. Data Management
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.
Figure 1. Chemotaxis of Human Jurkat T cells in response to SDF1α. Human Jurkat T cells (2.0x106 cell/ml) were exposed to a gradient of SDF-1α (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-1α, as evidenced by the increase in resistance and microtransients, while no movement was seen with exposure to RPMI 1640.
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).
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.
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 ¼" plexiglass, 2 mm apart (measured from inner edges). These sharpened cannulae are carefully inserted into agarose to cut the wells.
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.
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 evidence that overlapping or sequential gradients can influence cellular behaviors in novel ways. Moreover, it is likely that these more complex gradients are the norm in situ 12, 13.
The ECIS/Taxis assay can enable modeling of these different gradient configurations in ways that are not possible with other technologies. For example, by adding additional wells, one can distribute the orientation of the gradient(s) in relation to the cell well and target electrode. It is also possible to configure the assay to place cells in one of the wells as the source of chemotactic factors.
When setting up the assay, it is important to maintain complete hydration of the gel that overlays the target and counter electrodes in each chamber, and to cut wells that have the correct spatial relationship to the target electrode. Moreover, the bottom of the cell well needs to be contiguous with the thin film of liquid that forms under the agarose layer; it is in this film that the cells will move in response to the overlying gradient. To do this, one must aspirate all of the agarose plug without leaving gel fragments behind. The removal of the agarose plug cut by the cannula can create a tunnel between the two wells, which has the effect of deforming the gradient and allowing the cells to move freely across the target electrode. It is crucial that the vacuum pressure used to aspirate the gel plug is low to avoid compromising the agarose well integrity.
We have found that variations in the percentage of agarose used in the gel can differentiate cells that express cytoskeletal defects from wild-type cells, suggesting that this manipulation can be used to interrogate the forces that cells can exert on their environment. If the gel dehydrates during culture, changes will occur that influence the experimental results, including a relative increase in gel rigidity that would slow cell movement and an increase in solute concentration that would decrease total system impedance.
The assay is compatible with the measurement of movement by leukocytes and other cell types that do not allow current flow through the cell body. This is not a universal characteristic of cells: nerve cells and some epithelial cells (for example) could transmit current through the cell body and would thus produce far lower resistance values per individual cell.
While these approaches are specifically defined to detect cellular responses to chemotactic gradients, it is easy to imagine the assay as similarly applicable to studies involving metastasis and the extracellular signals that enhance metastatic cell movement. Moreover, there are additional array configurations that can be used in other cell-substrate interaction studies.
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.
This work was supported by grants from the National Institutes of Health (ES07408 and EB00208).
|ECIS Zθ||Applied Biophysics||www.biophysics.com/prodducts_Ecisz0.php|
|ECIS Electrode Array||Applied Biophysics||8W Chemotaxis||www.biophysics.com/cultureware.php|
|Seakem GTG agarose||BioWhittaker||50070|
|HyClone Fetal Bovine Serum||Thermo Fisher Scientific, Inc.||SH300703|
|Penicillin/Streptomycin||MP Biomedicals||1670049||Penicillin 5,000 IU/ml; Streptomycin 5 mg/ml|
|HEPES Buffer||MP Biomedicals||1688449||1M solution, cell culture grade|
|14 Gauge stainless steel Cannula (2) 4 inch||General Supply||5-8365-1||Blunt point|