This method can help understand key questions in the immune cell migration field, such as how the cytoskeleton sends shear forces and how integrin ligands and chemokines affect flow-induced migration of leukocytes. The main advantage of this technique is that it's a new type of migration assay that's easy for beginners to perform. It uses only commercially available equipment and reagents, as well as free software.
Though this method can provide insight into marginal zone B cells, it can also be applied to other immune cells, such as activated T cells. On the day before the experiment, thaw an aliquot of ICAM-1, and dilute it in PBS to a final concentration of five micrograms per milliliter. Then, add 30 microliters of ICAM-1 to the desired chambers of a flow slide.
Place the slide into a humid chamber, and set it at four degrees Celsius to incubate overnight. The next day, wash the chamber by adding 100 microliters of PBS to one well and then withdrawing 100 microliters from the other well of the chamber. Then, add 100 microliters of blocking buffer to a chamber, and incubate the slide in a humid chamber at room temperature for 90 minutes.
Next, wash the chamber by adding 100 microliters of PBS to one well, withdrawing it from the other well, and then add 100 microliters of migration buffer to the chamber. The slide is now ready to use. Store it in a humid chamber at room temperature until the experiment.
First, plug in a fluid pump, and attach the fluidics unit. Then, turn on the pump software. Next, wash the tubing once with five to 10 milliliters of migration buffer.
This will take about one minute. Then, add 11.7 milliliters of migration buffer equally to both reservoirs, and remove the air bubbles. Now, clamp the tubing about 10 centimeters from the connector piece, and place the fluidic unit in the heated incubation chamber on the microscope.
In the software, set the slide choice to micro-slide VI 0.4 and the tubing option to white. Also, set the desired shear stress to around four dynes per square centimeter and the imaging time to 30 minutes. Then, set the desired flow rate and shear stress by entering the values into the pump software.
Next, pre-warm the microscope incubation chamber, fluidics unit, and migration buffer aliquots to 37 degrees Celsius. Once warm, test the fluidic pump, ensuring that the migration buffer flows back and forth in the reservoirs and that there are no air bubbles in the system. First, isolate leukocytes, and purify out the marginal zone B cells as described in the accompanying text protocol.
Then, clean the bottom of the slide with an ethanol-dampened tissue. Add 30 microliters of the cell suspension into one well of the chamber, and withdraw 30 microliters of the stored migration buffer from the other well. Cover the slide, and incubate it for 30 minutes to allow the cells to attach.
Next, slowly add pre-warmed migration buffer to each well of the slide until a positive meniscus rises out of the well. Remove any air bubbles with the pipette tip. It is important to maintain consistency with factors that affect the cell adhesion, so keep the buffers and the cells at 37 degree, and avoid using excessive force when pipetting the buffer into the chamber.
Clamp the slide to the microscope stage, and remove the unmarked side of the fluidic tubing from the connector piece. Then, fill up the end of the tubing with migration buffer until a positive meniscus without air bubbles rises out of the end. Flip the end of the tubing over, and insert it into the top well of the flow chamber.
While keeping the tubing clamped, repeat the procedure for the marked end of the tubing. Now, switch the imaging system to Live mode, and select a field of view that is in the middle of the slide. Here, set the focus on the cells, and then de-focus slightly to enhance the black outline of the cells and produce a white interior.
The black background and white center will be easier to use in the automated tracking program. Set up the imaging sequence program to record one image every five seconds for 30 minutes using a 10x dry objective. Then, begin recording.
Remove the clamp from the tubing, and turn on the pump. This will cause the non-adherent cells to wash off and adherent cells to begin migrating. At the end of the imaging program, label and save the movie.
If using an inhibitor or a modifier of cell migration, it can be added to either the cells in the cell suspension before putting them on the slide or to the migration buffer in the fluidics unit. To begin automatic migration track analysis, open the ImageJ program. Copy the MTrack2_kt Java file to the ImageJ Plugins folder.
Under the Plugins menu, select Compile and Run. Then, restart ImageJ, and the plugin should appear in the Plugins menu. Open the image stack from the cell migration movie in ImageJ, and process the images as shown here in order to convert the video from color frames to high-contrast images showing the cells as black objects on a white background.
Next, sharpen the images twice, despeckle the images, and then convert the images to eight bit. In the Image menu, go to the Adjust Brightness/Contrast submenu, and put the Contrast slider at maximum contrast. This will make the cells appear as white objects on a black background.
Then, go back to the Adjust Threshold submenu to ensure that the cells appear as black objects on a white background. Now, run the automatic cell tracking plugin MTrack2 kt. On the options screen, set the particle size minimum to one pixel and the particle size maximum to 30 pixels.
Set the velocity value to 10 pixels, although marginal zone B cells will generally not exceed two pixels on successive frames that are five seconds apart. The cell tracks are now ready for analysis using the ibidi Chemotaxis Tool. These two fields show the migration paths of marginal zone B cells that were seeded on ICAM-1-coated slides.
The cells were tracked automatically with MTrack2 using the methods described in this article. On the left, cells were imaged under static conditions, and on the right cells were exposed to a shear flow of four dynes per square centimeter. The individual cell tracks can be imported into the ibidi Chemotaxis Tool to generate track plots of each movie.
Here, the individual cell tracks are colored red to indicate that they ended downstream of their origin point or black if they ended upstream. The analysis of cell tracks from four individual movies for both flow and no flow conditions are shown here. This includes the average directional migration index, cell velocity, path straightness, and the final straight-line distance for both conditions.
After watching this video, you should have a good understanding of how to image the directional migration of marginal zone B cells towards shear flow and how to automatically track the cells. During this procedure, other methods such as TIRF microscopy can be performed to answer additional questions, such as how does the cytoskeleton and integrins respond to shear stress?
