Cellular Impedance-Based Analysis for Monitoring of Cellular Processes in Real-Time

For the continuous measurement of cell behavior using real-time cell analysis, RTCA, obtain an RTCA microtiter plate containing impedance measuring-gold microelectrode arrays embedded in the well bottom. Carefully add cell culture medium to the wells.

Within the electrically conductive medium, apply a weak electrical potential across the microelectrodes. The potential causes electric current to flow between the microelectrodes, facilitating the measurement of the baseline impedance — the medium's inherent electrical resistance. 

Pipette the desired concentrations of mammalian cell suspension to the medium-containing wells. Allow the cells to uniformly distribute in the well.

During incubation, the cells adhere onto the well bottom.  The adherent cells on the microelectrodes act as an insulator, restricting the current flow and increasing the impedance.

Gradually, the cells proliferate on the microelectrodes, leading to increased exponential impedance.

Following confluent cell layer formation, the cells encounter nutrient deprivation and undergo death. As a result, the cells detach from the microelectrodes, decreasing the impedance.

The continuous impedance measurement facilitates real-time monitoring of the cellular events, including cell proliferation and death.

To determine the appropriate cell quantity for cell infection, prepare 24-hour, approximately 80% confluent MDCK cell cultures, as demonstrated, before washing the cells with PBS, and harvesting them with a 45-minute incubation in 3 milliliters of 0.25% trypsin-EDTA per flask at 37 degrees Celsius.

When the cells have detached, stop the reaction with 7 milliliters of fresh culture medium per flask, and count the cells from each culture. Dilute the cells to 4 x 10⁵ cells per milliliter of cell culture medium, and perform two-fold serial dilutions of the cells, as indicated.

Place an E-plate at room temperature for several minutes, before adding 100 microliters of cell culture medium to each well without touching the electrodes of the E-plate. Unlock the cradles and insert the plate front end into the cradle pocket of the impedance-measuring instrument. Close the door of the incubator, and open the software.

In the "Default experiment pattern setup," highlight the selected cradles and double-click on the top page to enter the name of the experiment. Click "Layout" and enter the necessary sample information for each selected well of the plate. Click "Schedule" | "Steps" |, and "Add a Step." The software will automatically add a 1-second step to measure the background impedance.

Click "Execute" and "Start / Continue." Click "Plot" and "Add" to select the appropriate wells, confirming that the background impedance is between negative 0.1 and 0.1, before proceeding to the next step.

Next, remove the plate from the cradle and add 100 microliters of each cell suspension to the appropriate wells in duplicate. Leave the E-plate in the laminar flow hood for 30 minutes at room temperature to allow a uniform distribution of the cells onto the bottoms of the wells, before loading the E-plate into the cradle pocket. Click "Schedule" and "Add Step," and enter values to monitor the cells every 30 minutes for 200 repetitions before selecting "Start / Continue."

To check and plot the cell impedance data, click "Plot" and select the concentration of cells that is just before the stationary phase 24 hours after seeding, to obtain cells that are still in a growing phase during the viral infection.