Here, we detail the experimental techniques used to evaluate the protrusion forces that podosomes apply on a compliant film, from the preparation of the film to the automated analysis of topographical images.
The overall goal of this procedure is to quantify protrusion forces using atomic force microscopy. This is accomplished by first preparing the formula coated grids and measuring the thickness of this film. The third step of this process consist in seating cells on the grids and leaving them to adhere.
The final step of the procedure is to image the forma film beneath the cells using atomic force microscopy. When adhering to substrate, microphages form submicrometric adhesion structures call podosomes. Ultimately, the deformation of the film in used by podosomes will be quantified and converted into forces using a homemade mechanical model.
Place an ethanol clean glass light into the funnel film casting device and cover the top of the funnel. Pan the fomvar solution up into the funnel until the level reaches two third of the slide. Leave it in for one minute then drain the formvar solution in a steady stream.
Note that the thickness of the film depends both on the stream rate and on the concentration of the formvar solution. Pat this light dry darkedly and cut out rectangles with a sharp razor blade. Dip the slide into your beaker filled with distilled water so that the fomvar film floats onto the water surface.
Carefully place acetone clean grids onto the films, shiny face up with a 12 millimeter diameter cover slip on top of some of them. They will be used for the thickness assessment experiment. Eventually dip a sticker covered slide to recover the whole film and grids.
Outline the glass covered slip with tweezers to cut the formvar and detach it from the slide. Mount the location of the grid beneath the covered slip then outline the grid with tweezers to pull it off. The hole in the fomvar film thus created will be imaged to measure its thickness.
Mount a silicon nitrate cont deliver in the AFM glass block making sure the golden stripe isn't beneath the tip of the spring. Mount the glass block onto the AFM module then place the latter onto the microscope stage. Place the fomvar coated cover slip on the observation chamber with PBS on top.
Scan the border between fomvar and glass in contact mode and with a 0.5 nano newton point. In the data processing software, use the glass surface as height reference, and measure fomvar thickness on the cross section. Under a sterile hood pick a 12 millimeter cover slip and stick a strip of the hole sided tape on it.
Outline the fomavr coated grid with tweezers and place it shiny face up so it will only it sides attach to the adhesive. This is to prevent fomvar from being torn when removing the grid from the tape. Place the cover strip into a sterile six well plate and pipe in a 10 millimeter droplet of serum free culture medium.
It should contain approximately 10 thousand cells and in this case, these are microphages differentiated from human monocytes. Make sure not to touch the grid with the piper tip in the process, so as not to damage the fomvar coating. Incubate for half an hour to let the cells adhere, then fill up the well with serum supplemented medium, and incubate again for two hours, before AFM observation.
Place two parallel strips of double sided tape on a glass bottom petri dish, making sure the width between them is slightly smaller, compared to your grid diameter. Then detach the grid previously seeded with cells and place it on top of the two tape strips with cells facing down. Fix the grid by placing two other strips on top of the pervious ones.
Fill the dish with preheated culture medium supplemented with hepe's. Place the dish into the dish heater previously set at 37 celsius degrees. Install the set on the AFM stage.
When the system temperature is stable at 37 celsius degrees proceed to the scan of the fomvar surface, which should be performed in the contact mode, with a 0.95 nano newton set point and visual the protozoan associated deformations on the deflection data view window. Here are the representative results and processing steps to yield formations with AFM to the graphical measurements. As you can see on this image, bulges on the fomvar surface are detected.
In order to convert this photographical information into force values it is necessary to first have access to the local high difference. To do so a third degree polynomial fit should be subtracted to its scan line independently. Then the use of a homemade dedicated imagery algorithm allows to evaluate protrusion force values from AFM height map an the mechanical model of protozoan mediated protrusion.
Thanks to this procedure, we are able to investigate the mechanisms involved in protrusion force generation at protozoans. Thank you for watching, and good luck for your experiments.
