The overall goal of this procedure is to measure chemotaxis in an automated real-time assay, which utilizes electric cell substrate impedance sensing, or EISs. This is accomplished by first preparing the axxis electrodes by filling each chamber with 0.5%agros in complete RPMI medium. The next step is to simultaneously cut two wells into the agros of each chamber with a 14 gauge cannula jig, and to aspirate out the two agros plugs in each chamber.
One well is filled with a single cell suspension while the other is filled with chemo attractant. Next, the electrode is connected to the ISIS theta system through the array holder that is placed in an incubator set to atmospheric parameters appropriate to the cell type being tested. Ultimately, cellular chemotaxis can be observed through measuring the change in resistance in real time as cells reach the target electrode between the two wells.
The main advantage of the axxis assay over existing methods such as Boyden chamber assay, or conventional microscope based under agros assays is that it allows for real-time automated measurements of chemotaxis without the need for ongoing manipulation of the system. To begin electrode preparation, stabilize the gold surface of the ISIS Texas electrode array by pre-treatment with sterile 10 millimolar cysteine in deionized water for 15 minutes at room temperature under sterile conditions. Following pre-treatment, aspirate the cystine solution from each electrode chamber.
Rinse three times with sterile deionized water and replace with 250 microliters of complete medium. Next, connect the electrode array to the contact pins on the instrument array holder to perform an electrode check. 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 repositioned in the array holder to establish contacts for all chambers. Once all chambers are connected, click on the check button in the ISYS Theta 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 five and six nanos for an eight chamber array. If an individual chamber is not within these acceptable parameters, it should not be used to collect data.
Next, disconnect the electrode from the array holder to prepare the individual aros chambers. Aspirate the medium from the chambers and wash each chamber three times with sterile deionized water. To begin setup of the aros chambers, prepare an aros solution containing RPMI medium as discussed in the written protocol.
Pipette 300 microliters of melted aros medium into each chamber, pipette the remaining agros into the cap of a 15 milliliter tube. To assess agros jelling, replace the ISIS axxis array cover and allow to gel at room temperature to build the reusable cannula. Well cutting tool sharpen two 14 gauge blunt end cannula.
Using a five 64th inch bit, rotate the tip of the drill bit in each cannula opening with a slow speed drill, making certain that the drill bit cuts the entire interior edge of each cannula opening. To form a sharp cutting edge drill two holes through plexiglass with a five 64th inch bit. Using a drill press insert each sharpened 14 gauge cannula into one of the holes so that they each protrude in equal distance from the plexiglass.
Sterilize the 14 gauge cannula tool by lightly flaming the cannula tips prior to cutting wells in the EIS chamber. Cool the cannula by touching the agros in the cap of the 15 milliliter tube. If the temperature of the cannula is not cooled, the aros will melt as the chamber wells are cut, rendering the wells misshapen and defective.
Cutting the wells without compromising the integrity of the agros is the most difficult aspect of this procedure. This requires careful alignment of the cannula and very low vacuum pressure. Align the cannula pair above the two gold dots surrounding the target electrode in each chamber.
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 using a sterile five inch pasture pipette connected to low vacuum pressure with a vacuum trap. To catch the agros waste, gently aspirate the aros plugs left by the cannula tool.
Then add 300 microliters of complete RPMI medium to the surface of each chamber at 37 degrees Celsius for two minutes to saturate the aros. After two minutes, gently aspirate the medium from the surface and the wells without disturbing the aros. Each well is capable of holding a volume of seven microliters to one well in each chamber and seven microliters of cell suspension.
In this demonstration, jca T cells are used at two times 10 to the seven cells per milliliter. Then load seven microliters of the chemo attractant into the second well of the chambers. For JCA T cells, 200 nanograms per milliliter of sdf one alpha diluted in incomplete.
RPMI is used as the chemo attractant for a negative control load. Seven microliters of complete media alone into the second well. 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 within the confines of the wells boundary. If cells are seen in both wells or are spreading beyond the cell well border, then a gap is formed under the aros. Note, the improperly set up wells as data cannot be reliably interpreted from those chambers.
To begin data collection, place the electrode in the array holder. Reconnect the gold pads on the electrode array to the spring loaded pogo pins of the holder click set up in the ISIS theta software. The wells that are properly connected will be highlighted in green.
If the wells are red, they're not connected, and the electrode should be repositioned in the array holder from the dropdown menu. In the software panel, select eight W one semicolon. This indicates the array configuration.Eight.
Well one electrode in the software control panel select check to determine the initial resistance and capacitance values. For each, well select multiple frequencies to measure resistance at several frequencies. Collecting multiple frequency data allows for analysis of capacitance, impedance, and resistance.
There is a choice of customizing frequencies or using a set of recommended frequencies. The standard multiple frequency acquisition settings will capture data at 11 preset frequencies at a rate of eight wells per minute. The time interval between measurements can also be customized.
Total experiment duration can be set on the right hand panel. Alternatively, the experiment can be stopped manually by clicking finish. Once cells have produced resistance that are indicative of arrival at the electrode, click start to initiate data collection.
Although the data can be exported to excel, the EISs 1600 R software that accompanies the EISs theta system is the most efficient method to analyze EISs taxes data. The software data analysis toolbar 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, which is elapsed time. EISs taxes data is most efficiently displayed as resistance over time at 4, 000 hertz.
Rapid fluctuations in resistance or micro transient are evidence of cellular movement over the electrode compiling or bending of the data points so that not all points are graft is not recommended for esys axis since it will average micro transient out of the data. Once the graph has been made. With the ISIS 1600 R software, it can be exported by choosing export graph from the file menu and saved as a or JPEG file.
Human jerk CAT T cells were exposed to a gradient of SDF one alpha to stimulate movement or to RPMI 1640 as a negative control. The normalized resistance at 4, 000 hertz was graft here. Cellular chemotaxis is indicated by an increase in resistance at 4, 000 hertz as human J CAT T cells move in response to SDF one alpha.
The arrival of cells on the target electrode is indicated by the appearance of rapid fluctuations in resistance called micro transients. The increase in resistance is directly proportional to the number of cells that cross the electrode. A negative result is indicated by stable resistance or by a slight decrease in resistance at 4, 000 hertz.
This is observed when the cells were exposed to RPMI 1640. The absence of micro transient is also a sign of an absence of cell movement at the electrode. In addition to this basic procedure, other modifications can be made to use Esys, Texas to monitor cellular movement in the presence of monoclonal antibodies specific for a chemo attractant, pharmaceutical modifiers, or toxins.
