A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

The overall goal of this procedure is to provide a simplified system for the analysis of cell durex through which many mammalian cells preferentially migrate towards a more rigid substrate. This is accomplished by first preparing Polymethyl suboxane or PDMS substrates of defined rigidity. Next, the prepared PDMS is poured into each well of a six well plate and any air bubbles are removed.

The Durex chambers are then completed by a placement of a sterilized cover slip on top of the PDMS in each well to create an overlay. The final step is to seed cells into the Duro taxes chambers at a density that gives the cells enough room to migrate without significant interactions with other cells. Ultimately, live cell microscopy is used to study axxis in migrating cells.

The main advantage of this technique over existing methods like poly acrylamide, is that there is a single step between the soft PDMS and rigid glass instead of gradient gel alternatives. Also coating PDMS with extracellular matrix Components like fibronectin do not require chemical cross-linking, which is required for poly acrylamide gels. To prepare one six well tissue culture plate first, tear the balance with a 50 milliliter conical tube.

Weigh out approximately 10 grams of the poly dimethyl suboxane or PDMS base solution in the 50 milliliter tube. For a 90 to one substrate, divide the measured weight of the PDMS base solution by 90. To determine the correct amount of crosslinker solution needed, add the calculated amount of PDMS crosslinker solution to the tube vigorously mixed A-P-D-M-S base crosslinker mixture for five minutes at room temperature.

Using a small spatula at this stage, the mixture will contain a small number of air bubbles. Centrifuge the PDMS substrate in a benchtop centrifuge for five minutes at 50 times. G.Add room temperature to remove the bubbles if there are still bubbles.

After the five minutes centrifuge again pipette one milliliter of the 90 to one PDMS substrate into each well of the tissue culture treated. Six well plate. Any remaining air bubbles present in the PDMS can be eliminated at this stage by popping them with a 21 gauge needle.

Allow the PDMS to spread for 30 minutes in the well. Next boil 12 millimeter glass Number one, cover slips in distilled water for five minutes, a total of three times. Place one dried cover slip into each well of the tissue culture plate by gently touching one side of the cover.

Slip into the PDMS solution and then dropping the cover slip onto the PDMS. As the cover slip settles, the PDMS will begin to encroach over the edges of the cover slip, but will not completely cover it. This will generate an interface between the PDMS and glass after curing.

Incubate the plate at 70 degrees Celsius in an oven for 16 hours to cure the PDMS. Finally, place the plate in a cell culture hood and UV sterilize for 10 minutes. Coat each durox chamber with one milliliter of fibronectin in phosphate buffered saline without calcium and magnesium for one hour at 30 degrees Celsius.

Make sure the entire surface is submerged in the fibronectin in PBS solution. Prepare heat natured 1%bovine serum albumin or BSA by weighing 0.5 grams BSA and dissolving it in 50 milliliters of PBS filter. Sterilize a solution through a point 22 micron filter before heating at 80 degrees Celsius for 12 minutes.

Next, aspirate the fibronectin solution and wash the chambers three times with PBS. Add one milliliter of heat denatured BSA in PBS to each well and incubate for 30 minutes at room temperature, trypsin eyes, and count the cells of choice while the durex chambers are blocking with the heat. Denatured BSA aspirate BSA solution from chambers just prior to plating cells.

Plate 10, 000 cells in a volume of two milliliters into each well of the doax chamber. Using the medium required for the particular cell type of choice, allow the cells to adhere and spread on the substrate for four hours in a humidified incubator at 37 degrees Celsius with 5%CO2, perform live cell imaging on an inverted microscope using phase contrast with a 10 x objective. The microscope should be fitted with an enclosed environmental humidified chamber, allowing control of temperature at 37 degrees Celsius and 5%CO2 during long-term imaging.

Imaging, after the cells have spread for approximately 3.5 hours, assemble the plate in the microscope chamber. Allow the samples to equilibrate in the chamber for 30 minutes. Set up automated multi-point visiting on the microscope if available.

Focus on the interface between the PDMS and the glass cover slip and choose points to image all around the interface. With an average of 40 points per Durex chamber image, the cells every 10 minutes for up to 16 hours, the region of interest will appear as two lines with the outer line corresponding to the edge of the cover slip and the inner line corresponding to the actual interface between the PDMS and the cover slip. To analyze the data, generate a spreadsheet like the one shown in the text protocol.

Count the number of crossing events from the PDMS to the glass surface and vice versa from each movie generated. A crossing event is defined as the cell nucleus passing over the boundary between the PDMS and the glass in either direction. Record the number of crossing events in the appropriate column in the Excel spreadsheet to quantify multiple crossings.

Count the number of times the cell crossed the interface. That number should be recorded in the Excel column corresponding to the substrate on which the cell was located at the end of the movie. Repeat the analysis for every cell that crosses the interface in the movie.

Exclude cells that migrate out of the field of view During imaging. Calculate the percentage of cells that migrated from PDMS to the glass surface and therefore underwent Duro Texas. To do so.

Add the number of crossing events from soft to hard and the multiple crossing events that ended on hard and divide by the total number of crossing events. Calculate the percentage of multiple crossings by dividing the total of multiple crossings by the total number of crossings to begin to understand the molecular mechanism by which cells correspond to mechanical cues in their environment. Specific proteins can be knocked down using IRNA as shown here.

Around 70%of the controls irna treated cells exhibited axxis. In striking contrast, when CD gap was knocked down using RNAi, the cells had no preference as to whether they migrated onto the hard or soft substrate. Furthermore, the CD gap knockdown cells crossed the boundary multiple.

Whereas control irna treated cells generally only cross the boundary once representative tracks of control. Irna cells show that the cells generally migrate across the boundary once and preferentially stayed on the more rigid surface as compared to CD gap. Irna treated cells that did not demonstrate a preference for either rigidity and cross the boundary multiple times.

After watching this video, you should have a good understanding of how to prepare the PDMS substrates, assemble the duris chambers and quantify DUR taxes.