Impedance-Based Measurements of Brain Cancer Cell Invasion

Take a cell invasion and migration plate containing upper and lower chambers.

The upper chamber contains a microporous membrane with an impedance-measuring gold microelectrode array on its bottom. Coat the membrane with an extracellular matrix or ECM solution. 

Fill the lower chamber with high-serum media.

Then, add low-serum media to the upper chamber and incubate to acclimate to culture conditions.

Apply a weak electrical potential across the microelectrodes to measure the baseline impedance.

Seed brain cancer cells in the upper chamber and allow them to settle on the ECM. 

The high-serum media acts as a chemoattractant, guiding the cells’ movement toward it. 

The cells secrete proteases that degrade the ECM, enabling their movement through the membrane pores toward the chemoattractant.

As the cells migrate, they adhere to the microelectrodes, increasing cellular impedance.

Over time, more cells migrate and attach to the microelectrodes, further increasing cellular impedance, which reflects the invasive potential of these cells.

Five to six hours before the real-time cell analysis, place the real-time cell analysis system into the cell culture incubator. To set up an invasion assay, use reverse pipetting to place 50 microliters of DMEM supplemented with 0.1 microgram per milliliter of extracellular matrix gel into each well of the upper chamber of a cell invasion plate. 

Immediately after plating, remove 30 microliters of the extracellular matrix solution from each well, and place the plate into the cell culture incubator for four hours. To set up an impedance measurement program six hours before the measurement, replace the medium in all of the electroporated cell cultures with low-serum medium. Under the Layout tab in the associated impedance measurement software, select quadruplicate wells for each biological condition. 

Under the Schedule tab, set a one-time baseline measurement sweep step with a one-minute interval and set a second step to measure the cell impedance in respective individual cradles for the actual experiment. One hour before the start of the cell impedance measurement, add 160 microliters of medium, supplemented with 10% fetal bovine serum as the chemoattractant into the wells of the lower chamber of the cell invasion and migration plate. 

Fill the wells in the upper chamber with 50 microliters of low serum medium, and place the chambers into the cradle of the system. 

Click the Message tab in the software to determine whether all of the wells are recognized by the control unit. If the message displays as expected, the plate in the cradle is ready for the experiment. Then, place the completely packed plates into the cell culture incubator in the real-time cell analysis system cradle for one hour to acclimate the plate to the cell culture conditions. 

While the plates are equilibrating, harvest the glioblastoma cells as demonstrated, and resuspend the cells from each condition at an eight times 10 to the fifth cells per 800 microliters of low-serum medium concentration.

To measure the baseline pre-migration reading, at the end of the acclimatization, click the cradle Start button. After the baseline measurement has been acquired, transfer the migration and the invasion plates from their respective cradles into a biosafety cabinet. 

Reverse pipette 100 microliters of cells into quadruplicate upper chamber wells for each biological condition in the appropriate well of the cell invasion and migration plates as programmed in the control unit of the cradle. After seeding, keep the plates in the biosafety cabinet for 30 minutes at room temperature to allow the cells to settle evenly on the plate bottom before transferring the plates to their respective cradles. 

Click the cradle Start button to begin measuring the cell impedance. To visualize the changes in cell impedance as the cell index in a time-dependent manner during or after completion of the experiment, open the Data Analysis tab. To visualize the data for each of the respective conditions, either individually or as averages and/or standard deviations, click the Option boxes for average and standard deviation. 

To export the cell index data to a spreadsheet file, place the cursor in the middle of the Data Analysis window and right click, in the dialog box that appears select the Copy Data into List Format option and paste the data into an open spreadsheet. To release the experiment, click the Release button in each cradle.