The overall goal of the following experiment is to quantitatively measure the migration of cells in real time using video time lapse microscopy. This is achieved by first coating a six well culture plate with collagen, and then sparsely seeding the cells of interest onto the coated plate. As a second step, a wound is created in the plate using a pipette tip, ensuring ample space for migration.
Next, the microscope is set up and images are captured at regular intervals using a temperature control system to allow continuous monitoring of migrating cells in real time. Ultimately, the particle tracking protocol is used to assess differences in the average speed and total displacement of test and control cells. The main advantage of this technique over existing methods like the avoidant chamber migration assay, is that it is not based on endpoint quantitative migration measurements in.
Instead, it allows monitoring and calculating different parameters continuously. This method can help answer key questions in the cancer biology field, such as how do particular genes or drugs affect tumor cell migration? Two days before the procedure add 1.5 milliliters of collagen diluted in opti media to each well of a six well culture plate, and then incubate the plate overnight at four degrees Celsius one day before the procedure.
Resuspend the cells of interest in two milliliters of DMEM plus FBS per well and transfer the cells into each well of the collagen ENC coated plate, incubating the cells overnight at 37 degrees Celsius. On the day of the procedure. Use a pipette tip to create a wound in the overnight cultured cells and wash each well with PBS to remove any debris that formed as a result of the wound.
Add DMEM plus FBS to the rinsed wells and then check under the microscope to confirm that a clean wound space has been created. After turning on the microscope camera and live cell imaging apparatus, position the plate over the microscope stage. Cover the plate by the new live cell environmental control chamber, and set the thermostat to 37 degrees Celsius and the carbon dioxide to 5%Now open the slide book software in the menu bar.
Select focus control by clicking the F within a circle button. In the objective box, use the dropdown window to define the objective In the filter set box, select settings to direct the light path to the I piece. Select the appropriate filter for Bright Field Illumination, and then click on open Bright.
To open the bright field shutter. Now select the bright field filter on the turret and view the sample through the eyepiece to find the appropriate fields and to bring the sample into focus. Then move the filter turret to change the light path to the camera For image capture in the slide book software, click on focus control select XY to select different positions through the I piece, and then click set point to lock in each location.
For image capture, manually fine tune the focus of the camera image that is viewed on the screen, and then use the tools in the focus controls window to adjust the Z position of the stage. For best results, focus on flat cellular protrusions near the contact with the plate to aid with focusing. Use the slider to adjust the exposure times to be in an appropriate range for focusing.
If multiple XY positions have been selected, adjust the focus for each individual position by selecting focus control. Then xy, then visit point. In slide book, select the camera graphic from the menu bar, select capture type, and then time lapse to select the length of time for the experiment.
From the dropdown menu, type in the desired delay between the beginning of one time point and the beginning of the next. In the interval fields, the third field will be automatically calculated from the manually entered values. Under multiple XY capture, select multi-point list for experiments with more than one XY position.
Finally, name the file to save the file, go to capture control and select advanced, then spool. Then capture to memory and save to spool file After each time point. Begin by clicking on image.
In the slide book menu bar, select import from the dropdown menu, and then click on slide book spool. Select the desired slide book files generated from the migration experiment for various fields and time points. Open the first file, then select mask on the menu bar and choose particle tracking protocol from the dropdown menu.
Under the particle tracking menu, choose manual particle tracking protocol. A window will appear with start and end time points To analyze random migration, select mask, and then particle tracking protocol. Again, to determine the coordinates for the center of the object, select center of area.
Then click track and continue. Now choose the desired statistics for each path such as displacement and average speed. As shown here, click calculate to view the statistics.
An example of a random migration analysis as quantified in terms of speed is shown in this box. In Whiskers graph, in this experiment, there was an increase in average speed in M-D-A-M-B 2 31 cells with a knocked down tumor suppressor gene. Compared to the control M-D-A-M-B 2 31 cells here, the random migration of the cells from the first figure was analyzed in terms of cell displacement.
Again, the total tumor suppressor gene knockdown group had an increased cell displacement compared to the control M-D-A-M-B 2 31. Cells in this time lapse video microscopy movie control MDA MB 2 31 cells were seated on collagen overnight. The images were then taken at one hour intervals for a total of 18 hours during random migration.
Note how the cells move away from their original positions over time. In this movie, the migration of test M-D-A-M-B 2 31 cells after overnight seeding on collagen was recorded and analyzed. Note that the test cells are more migratory compared to the control cells in the previous movie.
Following this procedure, other methods like cell invasion and dynas can be performed in order to answer additional questions like, what are the roles of cells in cancer metastasis After its development? This technique paved the way for researchers in the field of cell biology to explore cell migration using isolated cancer cell lines.
