A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

The overall goal of this procedure is to fabricate poly acrylamide or PA gels for cell culture with fluorescent microspheres embedded very near the gel surface. This is accomplished by first functionalizing glass cover slips with poly D lysine. The second step is to coat the glass cover slips with a solution containing fluorescent microspheres.

Next PA gels are formulated by mixing a appropriate concentrations of bis, acrylamide, and acrylamide, and sandwiched between an underlying surface and the functionalized side of the glass cover slips. The final step is to functionalize the gel with an extracellular matrix protein and culture cells on the gel surface. Ultimately, traction force microscopy is used to measure mechanical forces exerted by cells as a result of cell induced substrate deformation.

The main advantage of this technique over existing methods, such as traditional PHL fabrication, is that it localizes fluorescent microspheres to a known location within the gel depth, allowing for a more accurate calculation of cellular traction forces. Visual demonstration of this method is critical as spatial cure must be taken when functionalizing the glass cover slips in order to ensure the fluorescence bits to transfer properly to the PHL First place. Previously cleaned glass cover slips on a grated surface such that they are not touching.

To facilitate ease of interaction with the cover slips coat the entire surface of the cover slips with 0.1 milligrams per milliliter of poly D lysine for one hour. During this time, perform a one to 10, 000 dilution of the colloid solution of 0.1 micrometer diameter. Red fluorescent microspheres with deionized water to obtain a particle density of approximately one microsphere per 20 square microns on the gel surface.

Place the diluted solution in an ultrasonic water bath for 30 minutes. After one hour, use tweezers to carefully lift each cover slip and blow dry with air. Then return the dry cover slips to the grated surface.

Once the diluted colloid solution has been removed from the ultrasonic bath, pipette 150 microliters of the solution onto each cover slip. After 10 minutes, use tweezers to carefully lift each cover slip and blow dry with air. Once the dry cover slips have been returned to the graded surface, store them in the dark until ready to use.

Next, lay out the desired number of glass bottom Petri dishes on a flat surface in a chemical fume hood. Cover the glass portion of each Petri dish. Micro well with 97%of three amino propyl trimethyl for seven minutes for chemical activation.

After seven minutes, fill the Petri dish with deionized water and dispose of the solution in a waste container. Carefully cover the glass portion of each Petri dish. Well with a previously prepared 0.5%glutaraldehyde solution for 30 minutes.

After 30 minutes, fill the Petri dish with deionized water and dispose of it into a waste container to rinse and remove the glutaraldehyde hide. Before mixing the components of the poly Acrylamide gel solution, move the functionalized glass slides into the chemical fume hood such that they are easily accessible, allowing for the quick sandwiching of the gel with the glass bottom Petri dishes. After mixing the gel solution in a 15 milliliter centrifuge tube mix 40%bis acrylamide, 2%acrylamide and acrylic acid in immediate succession.

To achieve the desired matrix elasticity, then add 100 millimolar HEPs, 10%ammonium per sulfate, and teamed in quantities corresponding to desired matrix elasticity. To complete the gel solution, immediately pipette 15 microliters of gel solution onto the center of the glass portion of the Petri dishes. After picking up a functionalized glass cover slip with tweezers, flip it over such that the fluorescent beads are on the side making contact with the gel solution.

Then lay the cover slip gently on top of the now liquid poly acrylamide gel such that the functionalized side is in contact with the gel. Following this, flip all the Petri dishes over to assist with avoiding gravity effects on fluorescent nanoparticles. Polymerizing into lower levels of the poly acrylamide gel.

After polymerization, flip the Petri dishes back over and fill them with BS.Carefully make contact with the glass portion of the Petri dish and the outline of the cover slip using tweezers to scrape the circumference of the cover slip. Following several cycles of scraping. Remove the cover slip and dispose of it in a proper sharps waste container.

After removing all the cover slips, place the Petri dish lid on each dish and store at four degrees Celsius. Once the poly acrylamide gels have warm to room temperature, use a vacuum pump in a biological hood to remove all the PBS from the glass bottom dishes containing the gels pipette previously prepared soak solution onto each gel such that the gel is completely submerged. Following incubation at room temperature for at least one hour, add 150 to 250 microliters of a previously prepared of H-S-E-D-C solution to cover the gel surface and fill the glass bottom well of the Petri dish.

Then incubate the gels at room temperature for 30 minutes in the dark. Once the N-H-S-E-D-C solution has been removed by vacuum, add 150 microliters of a previously prepared fibronectin solution to each gel. Once again, incubate at room temperature for 35 minutes to allow for attachment of fibronectin.

At this point, warm cell media PBS and trypsin to 37 degrees Celsius in a water bath. Rinse the gels five times with sterile deionized water. Aspirating the water in between rinses, cover the gels, leaving them in the hood.

Add one milliliter of trypsin per 25 square centimeters to the flask containing the cells. After the cells are lifted from the flask, dilute the trypsin with cell media. Then count the cells using a hemo cytometer based on the gel surface area.

Determine the number of cells required per gel for a final cell seating density of 3000 cells per square centimeter dilute or concentrate the suspension such that 150 microliters of the cell media mixture contains this number of cells. Then Eloqua one 50 microliters of the cell suspension onto each gel. Following this place, the Petri dishes containing cells in an incubator for 30 minutes.

After carefully removing the Petri dishes from the incubator, fill the remainder of the Petri dish with approximately two milliliters of media such that the surface of the dish is completely submerged. Place the Petri dishes back in the incubator until image acquisition. When prepared for imaging, place one of the Petri dishes gently on the microscope stage.

After removing the Petri dish lid for DIC imaging, locate a single cell and capture a single still image of the cell in DIC without moving the microscope stage. Switch the imaging mode to fluorescence. Focus on the fluorescent microspheres and record an image of the microspheres.

Carefully remove the cell media from the Petri dish with the pipette and add 0.05%trypsin EDTA. Focus on the DIC image to show the gel from which the cell has now detached. Then image the microspheres under the cell after the cells have detached.

Confocal imaging was used to determine that the beads were underneath the gel surface and to quantify their precise location, fluorescent beads of a different size and wavelength than those inside the gel were allowed to settle on the surface. The location of the settled beads serves as a reference for the top surface of the gel where cells apply force upon adherence. Following confocal imaging, the distance between the embedded fluorescent nanoparticles and those on the surface was computed.

This distance determined the precise depth of the beads within the gel. Shown here is the bead spatial distribution and projected view on the YZ plane. The ladder provides the depths of all the beads from the gel surface.

The average depth of 0.1 micron beads is 619 nanometers 467 nanometers 278 nanometers for poly acrylamide gels of stiffness one 10 and 40 kilopascals respectively. The corresponding depths for one micron diameter beads are minus 20 nanometers in 40 kilopascal gels, 12 nanometers in 10 gels and 1, 255 nanometers in one kilopascal gels. The depth of beads dispersed throughout the gel was measured and compared to the depths.

Using the technique presented. The bead dispersion variability within the depth of a poly acrylamide gel using traditional fabrication methods is shown here. The displacement field for a fibroblast cell and corresponding fluorescent beads layer are shown here.

Beads near the periphery of the image appears slightly out of focus, which is an optical effect due to the water immersion objective. Upon addition of trypsin, the cell is released from the surface resulting in the deformed poly acrylamide gel surface returning to its original state. Once mastered, this PHL fabrication and functionalization technique can be done in six hours if it is perform properly.

After watching this video, you should have a good understanding of how to fabricate poly acrylamide gels with fluorescent microspheres, localized at a known location within the gel depth near the cell culture surface. For a more accurate calculation of cell traction forces.