Using this protocol, cell migration and invasion can be unveiled under real-time conditions enabling cell kinetics under loss or gain of gene function and drugs to be determined. Unlike other methods, no staining, mechanical cell damage, or fluorescent markers needed. Most importantly, the real-time cell kinetics can be determined in an effective manner.
When the U-118MG cell line culture reaches 70 to 80%confluency, wash the cells with PBS before treating the cells with two milliliters of 0.5%trypsin-EDTA. After 30 seconds at 37 degrees Celsius, stop the reaction with 10 milliliters of fresh culture medium and transfer the detached cells to a 15 milliliter conical tube. After counting, collect the cells by centrifugation and resuspend the pellet in a six times 10 to the fifth cells per condition concentration in the appropriate volume of fresh medium.
Transfer six times 10 to the fifth cells into one microcentrifuge tube per condition and add 800 microliters of Dulbecco's PBS to each tube. After centrifugation, resuspend each pellet in 60 microliters of resuspension buffer R and six micromolar of the siRNA of interest. Mix each tube with gentle tapping before electroporating 10 microliter aliquots of each cell suspension with three 10-millisecond 1, 350 volt pulses.
After each treatment, pool the electroporated cells from each condition in individual five milliliter aliquots of fresh culture medium. Then seed the cells into two 35 millimeter culture dishes per condition and place the cells in the cell culture incubator for three days. 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.
To measure invasion, assemble the upper chamber containing the extracellular matrix gel coated wells. To measure migration, use uncoated wells in the upper chamber. For either type of experiment, 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. Additionally, set aside a three times 10 to the fifth cells in two milliliters of culture medium for seeding in a 35 millimeter dish for downstream Western blot analysis. 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 dialogue 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.
Crk adapter protein knockdown decreases Crk1 and Crk2 protein levels by 85%and 86%respectively without inducing Crk-like protein levels. The knockdown of Crk-like reduces Crk-like protein expression by 85%with slight reductions in Crk1 and Crk2 protein levels. Combining both siRNAs reduces Crk1, Crk2, and Crk-like expression by over 80%while neither Vinculin nor alpha-Tubulin expression is affected by any combination of Crk or Crk-like knockdown.
Human brain glioblastoma cells migrate along a gradient in response to high serum concentrations reaching the maximum level of migration at 13 hours. In Crk knockdown cells, although the migration is initially delayed, the cells continued to migrate until 23 hours. Crk-like knockdown substantially reduces cell migration with the glioblastoma cell line completely losing their migratory ability upon knockdown of both Crk and Crk-like suggesting that Crk and Crk-like play essential overlapping roles in cancer cell migration.
However, when cell invasion is monitored over several days, the Crk knockdown cells reach a similar maximum level of cell invasion compared to control glioblastoma cells at 60 hours with Crk-like cells partially recovering their invasive ability by 90 hours and double Crk-Crk cells demonstrating a reduced ability to invade after 40 hours. The results clearly support the suggestion that cell invasion should be analyzed over the entire period of the experiment. Seeding the right number of cells and avoiding the air bubbles while recording the extracellular matrix for invasion assay and seeding cells are key points for success of this experiment.
Following similar procedure but using e-plates, cell adhesion and cell proliferation kinetics can be determined.
