The overall goal of this procedure is to create compliant matrixes for modulating and quantifying cellular contraction. This is accomplished by first activating a cover slip to allow Anchorage of the matrix. The second step of the procedure is to polymerize a poly acrylamide gel to the activated cover slip.
The third step of the procedure is to plate cells onto the gel and allow them to adhere. The final step of the procedure is imaging the gel to generate data to be analyzed by traction force microscopy protocols. Ultimately, results can be obtained that show cells applying traction forces to their matrix through traction force microscopy.
Hi, I'm Yvonne Eons from the lab of Margaret ELL at the physics Department and Institute for Biophysical Dynamics at the University of Chicago. I'm Steven Winter also from the Guard lab. Today we'd like to show you a procedure for making compliant matrices for measuring cellular contraction.
We use these procedures in our lab to study contractile actin mycin networks. So let's get started. Prior to cover slip activation, placed several 22 by 40 millimeter number 1.5 cover slips previously.
Rinse with 70%ethanol in a stainless steel rack such that cover slips are spaced apart and not touching. Working in a chemical fume hood, prepare 2%three or minor profile trimeth solution in isopropanol inside a square glass dish. Use a glass pasta pipette to add the three or minoro profile.
Trimeth oxy due to reactivity with plastic fully immerse the cover slips into this solution and incubate for 10 minutes while stirring on a stir plate in the fume hood. After soaking the cover slips, wash them by immersing in double distilled water. Exchange the water out four times after the final exchange.
Allow 10 minutes of soaking time with stirring. Then dry the cover slips in an incubator at approximately 37 degrees Celsius for 10 minutes. Once dry, allow them to cool to room temperature.
Immerse the cover slips in 1%Glutaraldehyde solution in double distilled water. In a glass square dish on a stir plate for 30 minutes. Make sure to use a clean dish.
Otherwise, the solution may appear cloudy afterwards. Wash the cover slips by three exchanges of double distilled water for 10 minutes per exchange with stirring. Next, cover the cover slips with aluminum foil to avoid dust and dry them at room temperature.
Once dry store the cover slips in a dry place away from dust for up to two months. To prepare the poly acrylamide or PAA gel, first make stock solutions of Acrylamide bis. Acrylamide Mix from 40%acrylamide and 2%bis acrylamide as described in the RI protocol.
DGAs the acrylamide solution in a vacuum chamber for 20 minutes. This reduces oxygen within the gel, which prevents polymerization. Meanwhile, prepare a 10%ammonium per sulfate or a PS solution.
Then wipe one by three inch microscope glass slides with rain X wipes to make the glass slide surface hydrophobic. Remove any dust with a Kim wipe to ensure a smooth gel surface. Cover the slide with aluminum foil and set aside.
Then remove the acrylamide solution from the vacuum chamber and add fluorescent beads. To initiate gel polymerization, add 0.75 microliters of TM E and 2.5 microliters of 10%a PS.Briefly mix the solution by pipetting up and down. Next, apply 10 to 12 microliters of the acrylamide solution to the previously prepared hydrophobic microscope Slide.
Place an activated 22 by 40 millimeter cover slip on top of the droplet. The gel solution should coat the entire cover slip. Smooth out any bubbles that may appear within the solution.
Allow the gel solution to polymerize at room temperature for about 10 minutes. Once polymerized, the gel will pull away from the edge of the cover slip. Using the fine tip of a pair of tweezers, carefully remove the cover slip from the microscope slide surface with the gel attached.
Immerse the gel in a Petri dish containing double distilled water To maintain a hydrated gel surface to cross link the extracellular matrix or ECM protein to the gel. First, prepare 40 microliter working aliquots of sulfur san par by dissolving SULF san par powder in anhydrous dimethyl sulf oxide or DMSO. Next, remove the double distilled water from the gel surface by briefly using our cover slip spinner.
Taking care not to dry the gel immediately before use. Dilute the sulfur sampa DMSO aliquots in double distilled water and coat the center of the gel surface with about 200 microliters. Then expose the gel surface to UV light in a UV crosslinker oven.
Following UV exposure, dip the cover slips in a beaker with fresh double distilled water. Remove any solution from the gel surface using a cover slip spinner. Then place param in a Petri dish container by a pet 50 microliters of cold fibronectin onto the param.
Invert the cover slip and place the gel side on top of the fibronectin. Allow the cover slips to react at room temperature for one to two hours or at four degrees Celsius overnight. Following incubation in a sterile tissue culture ventilated hood.
Place the cover slips in six centimeter tissue culture dishes containing PBS to coat the cover slips. Wash the cover slips extensively with several exchanges of PBS under sterile conditions following the wash. Sterilize the cover slips by UV treatment for 30 minutes.
Afterwards, incubate the cover slips in cell media for 30 to 45 minutes prior to plating cells. Next loader, 22 by 30 millimeter cover slip onto the top cover slip holder of a Warner Instruments confocal imaging chamber using vacuum grease to keep the cover slip in place. Place a chamber forming rubber gasket on top of the cover slip, allowing access to both inlet and outlet polyethylene tubing.
This will allow a spacing of 150 to 1000 microns between the top cover slip and the gel coated 22 by 40 millimeter cover slip to depending on the size of gasket used. Then load syringes containing warm cell media onto the inlet tubing via the connector kit, and check that the media flows through the tubing and onto the top cover slip. Apply vacuum grease onto the base of the chamber and load a gel coated 22 by 40 millimeter cover slip cell side up.
Apply the warm media to the cells. Place the top cover slip holder onto the chamber base with the chamber gaskets separating it from the gel coated cover slip. Make sure that the locating pins within the chamber base sit within the locating holes in the top cover slip.
Apply the pressure plate to the chamber base and use the pressure plate wrench to screw in and secure the pressure plate. Check the flow of media through the tubing and chamber to monitor any potential leaks within the chamber. And to eliminate any media free zones on the cell surface.
Apply the confocal imaging chamber to the stage adapter situated within a microscope holder for imaging image, fluorescently labeled protein and fluorescent beads embedded within the gel substrate on a confocal fluorescent microscope to obtain an image of unstrained bead position within the gel perfuse. Trips in to detach cellular adhesions from the gel and take an image of the fluorescent beads in the same imaging field where the cellars are dared. When the protocol is followed correctly, the gel surface will be relatively flat and smooth with fluorescent beads embedded evenly throughout.
The ECM can also be visualized by fibronectin immunofluorescence. If measuring gel contraction at the location of focal adhesions, imaging of the gel and cells should be done at the confocal optical plane of focal adhesions. Here, focal adhesions are visualized in attraction force experiment in a human osteosarcoma or U2 OS cell marked by GFP pxi.
The contraction of a gel can be visualized by the displacement of embedded fluorescent beads Underlying focal adhesions. Comparison of strained beads in green and unstrained bead positions in red allows for the quantification of gel substrate displacement under contraction here is a representative result of a spread cell that becomes detached from the gel surface by trypsin. Here is a representative result when comparing strained and unstrained bead positions.
The use of sophisticated computational algorithms can yield traction stresses associated with bead displacement and elastic modulus of the gels. These stresses are visualized here with a heat scale map and with vectors where the warmer colors on the heat map and larger arrows denote stronger forces. We've just shown you how to make compliant matrices for measuring cellular contraction when doing this procedure.
It's important to plan ahead so the time sensitive steps may be carried out quickly. So that's it. Thanks for watching and good luck with your experiments.
