Electrotaxis

ELECTROSTATIO BOND ELECTROSTATIO SUREAOE POTENTIAL ELECTROSTRIOTION ELECTROTAXIS ELECTROVALENT BOND ELEMENTARY OHARGE ELEMENTARY REACTION Elementary reaction stoichiometry, MOLECULARITY CHEMICAL KINETICS UNIMOLECULAR BIMOLECULAR TRANSITION-STATE THEORY ELEMENTARY REACTION Element effect,... 

C. elegans can display behavioral responses to external stimuli, for example chemical gradients and electric fields, which are called chemotaxis and electrotaxis, respectively [53, 54], By using microfluidics, the range and conditions of the applied stimuli can be controlled in a precise, flexible and repeatable way, and the corresponding responses of large-scale C. elegans can be detected in real-time [36, 52, 55, 56]. 

Zoospores of the phytopathogenic oomycetes accumulate at the potential infection sites of host roots by chemotaxis [11,15-17]. Root released electrical currents (electrotaxis) are also found to participate with... 

Electrotaxis and Wound Healing Experimental Methods to Study Electric Fields as a Directional Signal for Cell Migration... 

Electrotactic migration is not (at least not exclusively) mediated by chemotaxis. When chemical gradients in an electric field are disrupted with a continuous flow of culture medium perpendicular to the electric field vector, cells respond just as well to the electric field (20). G-protein coupled receptor signaling is essential for chemotaxis of many types of cells (22). Dictyostelium discoidum cells with a Gfl subunit null mutation lose their chem-otactic response while maintaining motility (23). Nevertheless, G(3 null mutants still respond to applied electric fields by directional migration, albeit less robustly than wild-type cells (20, 24). Therefore, electrotaxis and chemotaxis can be decoupled. 

We describe here techniques for electrotaxis experiments, with specific methods for corneal epithelial cells and differentiated HL60 cells (20). We also include the principles we use for analysis of electrotaxis from timelapse images. 

Fig. 3. Electrotaxis chamber and electric circuitry, (a) Chamber constructed in a plastic tissue culture dish, viewed from above, (b) Side view showing DC power supply, Ag/AgCI electrodes and agar-salt bridges that carry the voltage from the beakers to the two small medium reservoirs at the end of elecfrofaxis chamber. (Modified from (26)).

Voltage (or volt/ohm) meter for measuring voltage strength in the electrotaxis chamber. 

For collecting protein samples for Western blot, we use a similar electrotaxis chamber but of larger dimension 64 mm x 25 mm X 0.2 mm. Caution should be taken to ensure that the chamber is of even depth in order to maintain a constant voltage gradient across the large chamber. 

Preparation of Corneal Epithelial Cells In the Electrotaxis Chamber... 

Centrifuge twice at 160 for 5 min and resuspend cells in DMEM to a cell density of 14-24 x 10 cells/ml. Cells are ready for seeding in the electrotaxis chamber. 

Place approximately 1 ml of cell suspension in the electrotaxis chamber. We use fewer than 1 x 10 cells per 6.4 cm x 1 cm chamber for single cell migration studies. 1-2 x lO cells/ chamber of the same size is used for monolayer wound healing experiments. 

Place a coverglass roof over the electrotaxis chamber and seal with silicone grease (or petroleum jelly, e.g. Vaseline) on each side, leaving the ends open (Fig. 3a) (rr Note 6). 

Seed cells onto a sterile no. 1.5 acid-washed glass coverslip with an electrotaxis chamber similar to Fig. 3. 

Depending on the purpose of the experiment, the electrotaxis chamber is placed on the microscope stage (for imaging cell migration) or simply on the lab bench for protein sample collection. If the chamber is placed on the microscope, align the chamber so that the cathode and the anode are on the left and the right so that the electric field lines run horizontally as viewed down the microscope and recorded in the imaging system. 

Place the agar bridges to connect the beakers to each end of the electrotaxis chamber. Two holes in the cell culture dish lid made by cutting through with a hot scalpel blade will minimize evaporation of culture medium during long experiments (Fig. 3b). 

Place two measuring electrodes in the medium reservoirs at the ends of the electrotaxis chamber. 

Adjust the orientation of the electrotaxis chamber so that it is aligned with the microscope stage with the anode and cathode at left and right, respectively, and so that the electric held flows horizontally as viewed down the microscope. This is an important step as the angle measurement will not be accurate if the chamber is not properly aligned. Make a note of the positions (left or right) of anode and cathode. 

Fig. 6. (a) Electric field-induced dual phosphorylation of ERK1/2. Cells were starved overnight in serum-free medium. Fresh medium wifh or wifhout f 0% serum was introduced in the electrotaxis chamber and cells exposed to f 50 mV/mm for the periods of time shown. Three independent experiments were done with similar results. (Modified from (21Ji. (b) Akt in keratinocytes and neutrophils is activated in an electric field. 

Culture, differentiate, and seed PHAkt-GFP HL60 cells in electrotaxis chamber as detailed in Subheading 3.1.2. 

Mount the electrotaxis chamber on a heated microscope stage set at 37°C. 

Glass bridges and coverslips for electrotaxis chamber and the greases are sterilized by autoclave before use. Other materials and equipment that contact the cultures need to be sterile. 

Good electrical connections are essential for the electrotaxis experiments. Air bubbles are easily trapped in the electrotaxis chamber or in the agar bridge. It is necessary to check carefully the connections before switching on the power. Short circuit due to overflow of the medium outside the chamber needs to be carefully avoided. 

V. and Devreotes, P. N. (2002) Genetic analysis of the role of G protein-coupled receptor signaling in electrotaxis. J. Cell Biol. 157,921-927. 

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