Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

The overall goal of this procedure is to create poly acrylamide gels that can be used in both invader, podia, and traction force assays to study the invasive and contractile properties of cancer cells. This is accomplished by first activating the 14 millimeter cover slips in the worlds of glass blossom dishes to ensure that the poly acrylamide gels are bound to the glass. Next poly acrylamide solutions for both assays are prepared to yield gels with different rigidities or mechanical properties.

Then the invaded podia assays are prepared with a thin layer of fluorescently labeled extracellular matrix to detect degradation. Finally, the assays are seeded with cancer cells for overnight incubation, followed by fixing and staining of the invader, podia plates, and live cell imaging of the traction force plates. Ultimately, fluorescence microscopy is used to image invader, podia and extracellular matrix degradation.

While fluorescence and wide field microscopy are used to image microspheres and cells respectively for traction force calculations, Demonstrating the procedure will be Rachel Jarrell, a research assistant from my laboratory To prepare glass cover slips for poly equi mite gels. Begin by using low lint wipes to clean 12 millimeter cover slips. Use tweezers to hold 12 millimeter cover slips and 35 millimeter glass bottom dishes containing 14 millimeter cover slips and pass them through a bun and burner flame.

Treat the micro wells with 200 microliters of 0.1 N sodium hydroxide for five minutes at room temperature. Then aspirate and air dry the micro wells for 30 minutes. Next, under a fume hood, use a glass pipette to apply 50 to 100 microliters of three amino prop profile, Trimeth oxy silene to the micro wells and incubate room temperature for 10 minutes.

Then use ultrapure water to wash the micro wells for 10 minutes until the three amino profile trimethyl becomes clear.After. Use a squeeze bottle filled with ultrapure water to rinse the micro wells twice. Add two milliliters of ultrapure water to the micro wells and place on a rocker at medium speed at room temperature for 10 minutes.

Then aspirate the micro wells and air dry them for 30 minutes. Add two milliliters of 0.5%glutaraldehyde solution to each micro well and rocket it room temperature for 30 minutes. Finally, after using ultrapure room temperature water to wash the wells three times for a total of 30 minutes.

Dry the micro wells at a steep angle for 30 minutes. Using the information from the table shown here. Prepare one milliliter solutions of soft, odd, and rigid poly acrylamide gels by first combining the acrylamide bis and ultrapure water.

After degassing the solutions for 15 minutes at 200, 215, and 230 microliters of one milligram per milliliter. Human plasma fibrin in ultrapure water to the soft, odd and rigid poly acrylamide solutions respectively to all three solutions. At one microliter of 10 milligrams per milliliter, acrylic acid and hydroxy CIN aide or NHS Ester, five microliters of 100 milligrams per milliliter of a PS and two microliters of TM me.

Use gentle pipetting to mix and avoid introducing bubbles. Next pipette, 8.48 microliters of one of the poly acrylamide solutions onto the center of each micro. Well, to yield a PAA of 75 microns in thickness, then use tweezers to gently lower the flamed side of the 12 millimeter cover slip onto the droplet.

In the micro well allow the sandwiched poly acrylamide solution to polymerize for 15 to 30 minutes. When the solution has polymerized, add two milliliters of one XPBS to each dish and use tweezers to remove the 12 millimeter cover Slips now with two milliliters of one XPBS. Wash the microwells three times at room temperature for five minutes each.

Using the guidelines in this table, repair poly acrylamide gels for traction force assays in a similar manner as just demonstrated with the following exceptions. After adding the fibronectin sonicate eight microliters of 200 nanometer red fluorescent microspheres for 30 seconds. To each poly acrylamide solution, add eight microliters of microspheres, followed by the N-H-S-A-P-S and TE carefully mixed before pipetting to microwells.

According to the text protocol, to prepare poly acrylamide gels for invader podia assays, eat a 1%gelatin, 1%sucrose solution to 37 degrees Celsius. Apply 150 microliters of gelatin solution onto the poly acrylamide gels and incubate for one minute. Then carefully hold the glass bottom dishes at a 45 degree angle and gently aspirate the solution.

Keep the micro wells at a 60 degree angle for 60 minutes to dry the remaining thin layer of gelatin on the polyamide gels. Next, add two milliliters of chilled 0.5%glutaraldehyde solution, and incubate the microwells on ice for 15 minutes. Then incubate at room temperature for an additional 30 minutes.

Use two milliliters of one XPBS to wash the micro wells three times for five minutes each. After removing the final wash, add two milliliters of sodium borohydride solution and incubate for one minute. Gently tap the dishes on the bench to remove bubbles that form on the surface of the gelatin.

After aspirating the liquid, wash the micro wells three more times. Next, use one XPBS to dilute a ZI labeled human plasma fibronectin solution to 50 micrograms per milliliter before spinning at 175, 000 times g and four degrees Celsius for 15 minutes. Add 150 microliters at the fitzy fibronectin to each micro well and incubate for 60 minutes in the dark.

After the incubation, tilt the dishes to a 45 degree angle to carefully aspirate the solution from the bottom of the wells. Finally, under a dark cell culture hood, use 70%ethanol to fill the glass bottom dishes and lids and incubate it room temperature for 10 minutes. Then wash three times before carrying out invader, podia and traction force assays.

According to the text protocol in the invader podia assay invader podia are identified by colocalization of actin and court actin at punctate structures within the cell body, both actively degrading and non degrading invader. Podia can be counted and are differentiated by whether they co localize with black areas lacking fluorescent signal In the fitzy labeled fibronectin. In the traction force assay or images are captured at each cellular location marked by stage position first phase, and stressed images are taken of all the cells of interest.

A bottom image of the poly acrylamide is also taken at each position in order to calculate hydrogel thickness. After removing the cells, a null image is taken at each mark stage. Position deformation in the poly acrylamide gels are calculated based on the change in microsphere positions between the stressed and null images.

These displacement and the mechanical properties of the poly acrylamide gels are then used to calculate traction forces. After watching this video, you should have a good understanding on how to make poly acrylamide gels for inva podia and traction force assays.