The overall goal of the following experiment is to use electric cell substrate impedance sensing known as eis to characterize cellular behaviors. This is achieved by growing cells on electrodes in an array and taking measurements in different modes to quantify endothelial barrier formation, maturation and function manipulations such as electrical wounding and adding stimulants are then made to test cell motility and barrier function. Results are obtained that describe cell attachment, proliferation, migration, detection of endothelial permeability, and assessment of cell, cell and cell substrate contacts based on esis.
This method can help to answer key questions in the cell biological field, such as providing online generation of quantitative data, characterizing cells in their natural confluence state in their standard cell culture conditions. Generally, individuals new to this method with struggle because the underlying theory appears complex and you need to be aware of several basic considerations before you start an experiment. To begin, the arrays must be cleaned and stabilized to prevent electrode drift, improve reproducibility, and increase the signal strength ratio.
Therefore, add 200 microliters of 10 millimolar EL ine to each well of an eight well array after 15 minutes At room temperature, remove the EL cystine with two ultrapure water washes, not phosphate buffers. Also, do not expose the electrodes to serum containing solutions before coating as they can interfere with protein absorption. Next, add 200 microliters of warmed 1%gelatin to each well and incubate the array for half an hour at 37 degrees Celsius.
To remove the gelatin, use ultrapure water and do not allow the electrode surfaces to dry out. Then fill the wells with 400 microliters of complete culture medium, which can now contain serum. Load the array into the holder.
Check the nine gold squares. They should contact the POGO pins. Carefully secure the array in place by hand, tightening the adjustment screw on the computer.
Open the measurement software. Press set up and then check under the collect data section to perform a quick impedance measurement of each well at the default frequency of 4, 000 hertz. The values will be stored in the comments section.
Make sure the check accurately picked the type of EISs array in use under the well configuration section on the array diagram. Red and green indicate the functionality of the connections. If cleaning and coding were successful, eight W one E sys arrays should register about five to six NANOFARAD and eight W 10 E arrays.
50 to 60 nanofarad baseline resistance before cell seeding is thus about 2000 ohms. To model the data using RB alpha and cm, start an MFT recording with the medium failed array. Take 15 minutes of cell-free data and finish the experiment to add the cells.
Now remove the array and seed 400 microliters of single cell suspension into each well. To study cell growth seeds, 10, 000 cells per square centimeter to start with a nearly confluent population seed, 60, 000 cells per square centimeter. After loading the array into the holder, under the well configuration section, select the wells to measure.
Then go to the collect data options and select a measurement mode for a time series at a fixed frequency select SFT, and choose the measurement frequency to measure electrode coverage and model RB and alpha select MFT and the device will automatically take measurements at all the available frequencies. Run the measurement on multi frequency mode whenever possible. This requires data at all available frequencies and thereby provides most insights.
For micro motion analysis, select RTC and adjust the sampling frequency. The standard temporal resolution is one hertz, but can be increased to track fast changes in impedance. The value can be increased to 25 hertz for the z theta.
To measure micro motion sequentially from many wells, select the help menu. Then choose show expert toolbar menu items and acquire and multi-well RTC. In the collect data section, be sure to specify the time limit in hours.
When using this setting, the software will prompt for the number of cycles after data acquisition is started. When collecting data using SFT or MFT, select the time interval between measurements specified in seconds. For maximum data acquisition, leave this option unchecked.
Now press start and specify where the data should be stored. The run is stopped by pressing finish. By pressing pause, the data acquisition will stop, but the experimental clock will continue running.
Now remove the array and under a laminar flow hood, manipulate the wells. After returning the array to the holder, click check connection to verify electrical connections and then resume experiment to continue collecting data. If the cells do not need to be sterile after manipulation, then a stimulus such as thrombin can be added directly to a well during data acquisition.
Such introduced variations can be marked on the experimental clock by pressing mark and adding a comment. Another option is to electrically wound the cells. The settings for this need some tweaking to get right to short wounding time and it's not effective too long, and you can damage the electrodes.
Just start with the default settings the software provides and go from there. Go to The wound electro parade setup section and enable wound. Now select the wells to wound under well configuration.
Only the checked wells will be wounded. Clicking activate brings up a pop-up window to prompt for wounding. After wounding, check that the signal drops to a value of an almost cell-free electrode.
If it didn't repeat the wounding generally to work with the data, use the export data option and select to Excel. RB and alpha modeling, however, can be done from within the software. From the MFT data, remember that modeling is only valid in confluence cell layers.
And don't forget to add the cell-free reference. Start by automatically selecting a cell-free reference using the find function under the frequency scan modeling and analyzing section. Accept the value with set.
Next press model to start the calculations. Don't be alarmed if this takes several minutes. During a typical experiment, cells pass from a growth phase to a plateau phase.
When they reach confluence, images are acquired directly from the electrode during this process. The next phase is the formation and maturation of the EC barrier. Electrical wounding is then used to study cell migration.
A characteristic drop of the signal to baseline is followed by the reestablishment of the confluence state. Finally, the response to stimuli is followed in real time. Here, the vasoactive agent thrombin was applied.
This caused cellular contraction and thereby a transient opening of small gaps in the barrier, which caused a drop in impedance resistance and capacitance measurements yield complimentary information on cell adhesion and growth resistance at the most sensitive measurement. Frequency represents quality and function of the cell barrier and takes into consideration the resistance towards paracellular and transcellular Current flow. When cells attach to the electrodes, they restrict current flow and capacitance drops proportionally.
This phase provides an overall measurement of electrode coverage and is best quantified when recording capacitance at a frequency higher than 40 kilohertz. Resistance gives a clear idea of how good cells can block the current flow and hence the quality of the cell barrier. Therefore, cells of different passages and different types have different resistances.
Our RB and alpha helps distinguish between cell cell and cell matrix adhesions. RB is the resistivity of cell cell contacts to the current flow, or an inverse measurement of permeability. Alpha is a measure for impedance contributions from the cell electrode junctions.
Both of the RB and alpha values can be calculated from within the ISIS software. Small fluctuations in the resistance signal can be due to subtle motions in the confluence cell layer or micro motion. They can be measured with one E arrays at the most sensitive frequency and analyzed by fast Fourier transformation within the EISs software manipulations that give insight into cellular behavior can be made with precision.
For example, it is possible to make a 250 micron electrical wound that will close within a few hours to study cell migration. Impedance spectroscopy is also well suited to analyzing the effect of added substances. As noted earlier, thrombin makes the cell barrier hyperpermeable by lowering basal cell tension with ARO kinase inhibitor, the effect of thrombin could be diminished While attempting this procedure, it's important to remember ESIS is extremely sensitive to changes in the cell environment like temperature, pH, medium depletion, and so forth.
After it's development. This technique paved the way for researchers in the field of ology to study the trends and effects of vasoactive agents on confluence cell cultures in real time.
