This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.
Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video – time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.
1. Preparation of Plate Coated with Collagen
2. Cell Preparation and Seeding on a 6-well Plate
Note: Use warm media and PBS to ensure optimal conditions for cell movement
3. Microscope Setup and Automated Image Acquisition
There are several imaging software packages that control the microscope for automated image acquisition. Here, we use Slidebook 5.0 imaging software and automatic acquisitions are performed on a Olympus microscope (Olympus IX81), coupled to an EM-Hamamatsu C9100 camera, which is excellent for live-cell applications where speed of acquisition and minimal phototoxicity is paramount. The system includes a stage top environmental control chamber (Neue Live Cell) and an automated XY stage controller (Prior Proscan). Images are acquired with a 10X UPLANFL Ph1/0.30 objective, in bright field mode, using a defined time interval. The following protocol describes the image acquisition.
4. Image Processing and Analysis of Migration: Calculation of Speed and Displacement
In this example, image processing is done by SlideBook5, 6 5.0 software. There are various other programs such as ImageJ7 for the image processing and analysis.
5. Representative Results
An example of a random migration analysis as quantified in terms of speed and displacement (Figure 6). After exporting the file to excel, data is plotted using a box and whiskers plot. Statistical analysis comparing total displacement and average speed between test and control is performed using Mann- Whitney test. The test sample shows an increase in average speed as compared to the control. The test sample shows an increase in total displacement as compared to the control.
Movie of control and test: Control MDA- MB-231 and test MDA-MB-231 cells were seeded on collagen overnight. Time- lapse video microscopy was performed by Olympus microscope, coupled to an EM-Hamamatsu camera. Images are taken at an interval of 1 hour for a total of 18 hours during random migration. Please see Movie 1 and Movie 2 of random migration for control and test samples.
Microscope setup:
Figure 1. Setting up multiple points for image capture.
Figure 2. Steps depicting capture control. Click here to view larger figure.
Figure 3. How to save multiple images.
Image processing and analysis of migration.
Figure 4a. Steps to import image for data analysis.
Figure 4b. Steps to analyze data using particle tracking protocol.
Figure 4c. Window depicting path length of tracked particles.
Steps of data analysis.
Figure 5a. Steps depicting parameters of particle tracking protocol.
Figure 5b. Selection of path statistics to be analyzed.
Figure 5c. Steps to create mask for automated tracking protocol.
Figure 6. Average speed (Figure 6a) and displacement (Figure 6b) is plotted in a whiskers plot. The test sample shows an increase of speed and displacement as compare to control.
Movies
Movie 1. Migration of control cells. Click here to view the movie.
Movie 2. Migration of test cells. Click here to view the movie.
Control MDA- MB-231 and test MDA-MB-231 cells were seeded sparsely on collagen for overnight. Time- lapse video microscopy was performed by Olympus microscope, coupled to an EM-Hamamatsu camera. Images are taken at an interval of 1hour for 18 hours during random migration. Please see the movies of random migration for control and test.
The real time random migration assay enables accurate, sensitive, analysis of cell migration parameters such as speed and displacement. This method is not restricted to end point measurement value, hence it is more quantitative. Since, cells are monitored in normal DMEM medium with serum; it reduces the deleterious effect of longer incubation in serum free media. It has been shown that the migration of cells depends on the formatting and breaking cell contacts with the substratum8, thus, this method could be used to study effect of different substrata on migration.
One of the critical steps involves proper control of temperature to 37°C and CO2 at 5% to ensure optimum migration. The assay permits flexibility to monitor migratory responses at desired time points; however, it may require optimizing the number of time points. For example, some cells having lower migration rates may have to be monitored for a longer time as compared to cells having higher migration rates.
The microenvironment of the tumor is one of the crucial factors in carcinogenesis. Cancer results from interaction of tumor cells with surrounding cells such as endothelial cells, stromal and inflammatory cells 9. This assay can be modified to study how migration of tumor cells are influenced by the microenvironment. One of the ways in which it can be achieved is by studying the migration of fluorescently labeled tumor cells grown in a mixed population of different cells such as stoma and endothelial cells. This method can also be used to study wound healing in real time.
Time-lapse microscopy could be modified to monitor cell invasion, cell division, apoptosis and lamellipodial dynamics and focal adhesion dynamics using higher resolution lenses.
One of the limitations of this method is that it does not allow monitoring of migration in vivo. Since there is no equipment available that can measure real time migration in the body, this type of assay is not suitable for in vivo migration. It also does not allow us to elucidate the biochemical machinery and signaling components that are important in cell migration. In order to address the signaling pathways, it is essential to perform biochemical experiments. See references for groups that have used this method for random migration 6, 10-14.
The authors have nothing to disclose.
The authors would like to thank Amanda Struckhoff for the initial help with the experiment. This work was supported by a grant from NIH 5RO1CA115706.
Name of the reagent | Company | Catalogue number | Comments |
DMEM | Thermo Scientific | SH30243.01 | |
FBS | Gemini Bio- Products | 100-106 | |
Opti-Mem | Invitrogen | 31985-070 | |
Collagen | BD | 354231 | |
Microscope with Live Cell chamber & Automated XY stage controller | Olympus | Olympus 1X81 | |
Environmental control chamber | Neue | Neue Live Cell Chamber | Ours has a custom built rectangular glass plate top for appropriate optics through the top of the chamber. The rectangular shape accommodates multi-well plates used in many of our experiments. |
Automated XY stage | Prior | Prior Proscan | This allows precise return to multiply selected XY positions during time lapse microscopy. |
Camera | Hamamatsu EM camera C9100 | ||
Software | Typically purchased through microscope vendor such as Olympus | Slide Book 5 |