There's growing appreciation for the impact of the mechanical microenvironment on cell signaling and migration. Necessitating the use of unique, experimental approaches to deliver mechanical stimulation to individual cell. This technique allows the dynamic manipulation of the mechanical micro-environment underneath a cell in real time.
Allowing the observation of mechanically regulated changes in cell migration behavior, and sub-cellular signaling events. Demonstrating the procedure with me will be Nyla Naim, a post-doc and Johnathan Patterson, a technician from our laboratory. Begin by using a Chorono-1 to activate the glass surface of each bottom cover slip for 20 seconds, before immediately over-laying 50 microliters of bind silane working solution onto each cover slip.
Allow the solution to dry for 10 minutes before rinsing the cover slips two times with 95%ethanol and two times with isopropanol. Then, allow the cover slips to air dry for approximately 20 minutes. For fluorescent micro-bead deposition, first sonicate the working micro-bead solution for one hour.
15 minutes before the end of the sonication, place the activated cover slips vertically in a ceramic cover slip holder and transfer the holder into a table-top plasma cleaner for room-air plasma treatment for three minutes. Next, place pieces of paraffin film in 60 milliliter Petri dish lids, and place the cover slips onto the films, lightly tapping each cover slip to ensure a good contact between the film and the coverslip. Then, add 150 microliters of the working bead solution to the top of each coverslip, and immediately aspirate the ethanol solution from the side of the coverslips, leaving the beads on the coverslip.
To cast the Hydrogels immediately after their preparation, add a 25 microliter droplet of Hydrogel solution to the activated side of each bottom coverslip, and immediately flip the beaded top coverslips, bead-side down, onto the droplet. Allow the gels to polymerize for 30 minutes before using forceps to gently remove the coverslips. Then, rinse the gels with three, five minute washes in fresh 50 miliMoler Hepes per wash.
To activate the Hydrogels surfaces, incubate the gels in fresh 50 miliMoler Hepes, supplemented with 0.4 miliMolar Sulfo Sanpah, and immediately expose the gels to an ultraviolet arch lamp in an enclosed area for 90 seconds. At the end of the activation, wash the gels with three, five minute washes in fresh Hepes, and treat the activated Hydrogels with 20 micrograms per milliliter of fibronectin for one hour at 37 degrees Celsius. At the end of the incubation, aspirate the excess fibronectin solution, and wash the gels three times in PBS for five minutes per wash.
After the last wash, place the Hydrogels in a small volume of PBS and sterilize the gels for 15 minutes under ultra-violet light in a tissue culture hood. Then, wash the gels one time in fresh PBS. For cell plating, seed 2.1 times 10 the four of the cells of interest in three milliliters of medium per-Hydrogel and allow the cells to adhere at 37 degrees Celsius for four to 18 hours.
While the cells are equilibrating, use a micro-pipette puller to pull a one millimeter diameter by 100 millimeter long borosilicate glass micro-pipette, to obtain a taper of over two millimeters that reduces to approximately 50 micrometers in diameter in the first millimeter, and extends to a long, parallel 10 micrometer diameter tube in the last millimeter. Next, load the pipette into a microforge and shape the pipette to have a 15 micrometer blunted tip that is enclosed at the very end of a 250 micrometer section, bent at approximately 35 degree angel from the rest of the pipette. The approximate diameter at the bend should be around 30 micrometers to lend strength to the tip.
Next, place a Hydrogel onto the stage of an inverted microscope. Cover the medium with mineral oil and select the 10x objective. Sterilize the prepared micro-pipette with the 70%alcohol.
Then, insert into the micro-pipette holder with the hook pointed toward the dish, and use the course manipulator to lower the pipette until the hook just touches the surface of the liquid. Focus the microscope on the cells adhered to the top of the gel, and taking care that the objective is in no danger of hitting the sample or stage, bring the focus above the gel to find the tip of the micro-pipette. Rotate the pipette until the tip is perpendicular to the focal plane, so that the blunted tip of the micro-pipette is pointing down, and refocus on the tip of the pipette as necessary.
Focus back down to the cell layer to gauge how far the pipette is from the gel surface, and focus back up to a plane that is part-way between the gel and the tip of pipette. Then, slowly lower the pipette to reach the intermediate focal plane, and increase the magnification to that which will be used in the experiment, before lowering the pipette until it hovers just above the surface of the Hydrogel. For micro-manipulator calibration, image an area devoid of cells, an area with beads, but no manipulation, with the pipette engaging the gel, and with the engaged pipette pulling the gel.
Then, use image J to compare the no bead fields to the bead fields without engagement, the bead fields with the gel engaged, and the pulled gel to calculate the relative bead displacements and force applied to the gels. To conduct a durotaxis assay, monitor a group of cells for 30 minutes to identify cells that are moving in a directed manner. Select a cell that is steadily moving in one direction, and monitor the cells of the desired frame-rate for an additional 30 minutes.
Next, position the pipette about 50 micrometers in front of the near side of the leading edge of the selected cell, and move the micro-manipulator such that the gel is deformed orthogonally to the cell's direction of travel. Then, observe the cell over time as it responds to the acute, local gradient of Hydrogel stiffness. Using this technique as demonstrated, rat embryonic fibroblasts move towards the increased stiffness in gradients applied by a glass micro-pipette.
Rat embryonic fibroblast cells, transiently expressing vinculin tension sensor on 125 kilopascal polyacrylamide gels also demonstrate the formation of focal adhesions in the directions of the stretch over a period of 40 minutes. Forster Resonance Energy Transfer analysis reveals that vinculin localized to focal adhesions experiences an immediate change in tension, when presented with an acute durotactic stimulation. Expanding the utility of this assay to the observation of sub-cellular signaling events, in response to durotactic stimulation.
If you make multiple attempts to stretch a cell, or if the microneedle slips during the assay, start over with a new cell to avoid imparting multiple, variable, mechanical stimuli. Beyond its utility for assaying durotaxis, this technique can be combined with genetic approaches, such as biosensors, RNAI, or gene editing to help delineate the molecular mechanisms involved in mechanotransduction.
