Impedance-Based Measurements of Brain Cancer Cell Invasion

Samenvatting

Source: Mudduluru, G., et. al. Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion. J. Vis. Exp. (2020)

This video demonstrates an impedance-based assay to measure glioblastoma cell invasion and migration. Cells degrade the extracellular matrix, move through microporous membranes, and adhere to electrodes, generating impedance. This real-time analysis reflects their invasive potential for cancer research applications.

Protocol

1. Preparation of the Real-time Cell Analysis System, Cell Invasion and Migration (CIM) Plates, and Electroporated U-118MG Cells for Plating

1. Place the real-time cell analysis system in a CO₂ incubator under culture conditions 5–6 h prior to the start of the experiment to stabilize the system to the culture conditions.
2. For the invasion assay, plate 50 µL of DMEM (plain medium) containing extracellular matrix (ECM) gel at 0.1 µg/µL in each well of the upper chamber of the CIM plate. To avoid having any air bubbles, use the reverse pipetting method. Immediately remove 30 µL of ECM gel, leaving 20 µL in the well.
3. After the ECM gel coating, keep the plate in the incubator under culture conditions for 4 h with its lid on. During the ECM gel coating and drying, take preventive measures to avoid direct contact with the electrodes of the upper chamber of the plates with the hands, the surfaces of the biosafety cabinet, or the CO₂ incubator.
4. To set up the impedance measurement program, double-click the associated software icon (see Table of Materials) to open the system software application (control unit). Each cradle has an individual window with different tabs to set the experimental conditions, the impedance measuring time interval and duration, and data analysis.
5. Under the Layout tab, set quadruplicate wells for each biological condition and set two-step cell impedance measurements under the Schedule tab. The first step is for a one-time baseline measurement (one sweep with a 1 min interval), and the second step is to measure the cell impedance in respective individual cradles for an actual experiment. For migration, use 145 sweeps with a 10 min interval, and for invasion, 577 sweeps with a 10 min interval, individually set) in respective individual cradles.
6. One hour before the start of the cell impedance measurement, add 160 µL of DMEM with 10% FBS as a chemoattractant to the wells of the lower chamber of the plate. Assemble either the upper chamber containing the ECM gel-coated wells or uncoated wells with the lower chamber to measure invasion and migration, respectively.
7. Fill the wells in the upper chamber with 50 µL of low serum medium and place them in the cradle of the system. Check whether all the wells are recognized by the control unit by clicking the Message tab. If the message displays as OK, the plate in the cradle is ready for the experiment.
8. Preincubate the completely packed plates in the incubator under culture conditions for 1 h prior to measurements in the real-time cell analysis system cradle, which is essential for acclimation of a packed plate to the cell culture conditions.
9. For harvesting cells treated with low serum medium, wash the cells growing in culture dishes with 1x PBS and add 2 mL of 0.05% trypsin-EDTA (ethylenediaminetetraacetic acid). Place in the incubator for 30 s and remove the trypsin-EDTA. Collect cells in the low serum medium into a 15 mL tube and count the cells using a handheld automated cell counter.
10. After counting, centrifuge cells at 100 x g for 5 min and discard the supernatant.
11. Resuspend 800,000 cells in 800 µL of low serum medium for the migration and invasion assays. Additionally, resuspend 300,000 cells in 2 mL of culture medium and seed in a 35 mm dish for Western blot analysis to confirm the regulated knockdown of Crk and CrkL.

2. Baseline Reading, Seeding of the Cells, and Cell Impedance Measurement and Visualization

1. Measure the baseline reading by clicking the cradle Start button. The baseline should be read after preincubating the plates for 1 h in the cradle of the real-time cell analysis system under culture conditions in the carbon dioxide incubator and before seeding the cells to the respective wells in the upper chamber.
   NOTE: Once the cradle Start button is clicked, the control unit will ask whether to save the experimental file. After the file is saved, the cradle analyzer measures the baseline cell impedance as set in the program initially and enters a pause mode until the cradle Start button is clicked again to measure cell impedance in the second step.
2. Take out both plates for migration and invasion from the cradles after the baseline measurement and keep them in the biosafety cabinet.
3. Seed 100 µL of cells (100,000 cells) in quadruplicates for each biological condition in the upper chamber of the CIM plate in the respective wells as programmed in the control unit of the cradle by reverse pipetting to avoid air bubbles.
4. After seeding, keep the plate under a biosafety cabinet for 30 min at room temperature to allow cells to evenly settle down to the bottom. Transfer the plate back to the respective cradle and click the cradle start button to start measuring cell impedance as programmed in the second step of 1.5.
5. After the last sweep, the experiment is finished, and the results are saved automatically.
6. Under the Data Analysis tab, visualize the changes in cell impedance as cell index in a time-dependent manner during or after the completion of the experiment. Each of the respective conditions of quadruplicates can be visualized either individually or as averages and/or standard deviations by clicking the Options boxes for average and standard deviation.
7. To export cell index data to a spreadsheet file, open an empty spreadsheet file, place the cursor in the middle of the Data Analysis window, and right-click. In the dialog box that appears, choose the option Copy Data into List Format, and paste the data into the open spreadsheet.
8. Adjust the time in the raw data to represent the actual start time of the cell impedance measurement. The second step's start time is set as zero.
   NOTE: The control unit has the option to obtain the cell index with or without normalization (i.e., normalized cell index) and visualize the results as a graphical presentation in a time-dependent manner. In this example, cell index data are exported without normalization for processing and graphical presentation.
9. Release the experiment by clicking the Release button in each cradle.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Biosafety cabinet	ThermoFisher Scientific	1300 Series Class II, Type A2
CIM plates	Cell Analysis Division of Agilent Technologies, Inc	5665825001	Cell invasion and migration plates
Dulbecco’s modified eagle’s medium (DMEM)	ATCC	302002	Culture medium used for cell culture
Dulbecco's phosphate-buffered saline (DPBS)	Gibco	21-031-CV	DPBS used to wash the cells
Fetal bovine serum (FBS)	Hyclone	SH30910.03
Heracell VIOS 160i carbon dioxide incubator	ThermoFisher Scientific	51030285	Carbon dioxide incubator
Matrigel	BD Bioscience	354234	Extracellular matrix gel
RTCA software	Cell Analysis Division of Agilent Technologies, Inc		Instrument used for experiment
Scepter	Millipore	C85360	Handheld automated cell counter
Trypsin-EDTA	Gibco	25300-054
U-118MG	ATCC	ATCC HTB15	Cell lines used for experiments
xCELLigence RTCA DP	Cell Analysis Division of Agilent Technologies, Inc	380601050