Eenvoudige-Polyacrylamide gebaseerde Multiwell Stiffness Assay voor de Studie van stijfheid-afhankelijke cel responsen

The overall goal of this procedure is to enable a quick, efficient, and inexpensive preparation of poly acrylamide gels in a multi-well plate format. This is accomplished by first sandwiching, the gel precursor solution between a hydrophobic coated glass plate and an acrylamide adhesive flexible plastic support. The second step is to peel the gel off the glass plate, which has covalently attached to the flexible plastic support upon polymerization and let it dry.

Next, the dry gel and the underlying flexible plastic support are cut into desired shapes and glued plastic side down to the well bottoms of a multi-well plate or any other cell culture vessel. The final step is to coat the multi-well plate assembled poly acrylamide gels with a cell adhesive coating, such as a monolayer of collagen type one. Ultimately microscopy, as well as other cell characterization techniques are used to observe the effect of the underlying poly acrylamide substrate.

In particular, its stiffness on cell behaviors. The main advantage of this method over existing methods is that allows handling of any stiffness and thickness of poly gels as well as their assembly in a multi-well plate format, in a low cost and time efficient way, not afforded by other methods. Demonstrating the procedure will be done as a student in my laboratory.

First, prepare the poly acrylamide gel precursor solution by mixing acrylamide, the crosslinker bis acrylamide, and deionized water dissolve Sul OPA in dimethyl sulfox at 50 milligrams per milliliter. Aliquot 20 microliters of the stock solution into 10 micro centrifuge tubes. Then flash, freeze the solutions in liquid nitrogen and store at negative 80 degrees Celsius.

Next, dissolve ammonium per sulfate in deionized water to achieve a final ammonium per sulfate concentration of 10%weight by volume. Eloqua 25 microliters of the ammonium per sulfate solution into 10 micro centrifuge tubes, and store it negative 20 degrees Celsius. Prepare a 0.2 milligrams per milliliter of collagen solution by diluting the collagen type one stock solution in one XPBS at pH 7.4.

All solutions are kept on ice until use. At this point, place a few drops of a hydrophobic solution on a glass plate and use tissue paper to spread across the surface. After allowing the plate to air dry, wipe with tissue paper again to even out the hydrophobic coating.

Cut a flexible plastic support to match the size of the hydrophobic coated glass plate. Then mark the hydrophobic side of the flexible plastic support by lightly scratching the surface with a sharp tool such as a scalpel. For the gel preparation add 4972.5 microliters of poly acrylamide precursor solution of desired final concentration into a 50 milliliter conical tube.

Place the tube in a degassing chamber for 30 minutes with the cap opened. Following this, add 25 microliters of the previously prepared ammonium per sulfate solution to the DGAs gel solution to achieve a final ammonium per sulfate concentration of 0.05%Then add 2.5 microliters of TM me to achieve a final TM ME concentration of 0.5%Mix the solution gently by pipetting up and down three to five times. Next place silicone spacers of desired thickness onto the hydrophilic side of the flexible plastic support.

Then pipette the gel solution onto the flexible plastic support between the spacers and sandwich with a hydrophobic coated glass slide light. After allowing the gel to polymerize for 45 minutes, peel off the flexible plastic support with the covalently attached poly acrylamide gel on top and set gel side up to air dry. Once dried, cut the poly acrylamide gel into the desired shapes.

Prepare approximately 500 microliters of polymethyl soane per 96 Well plate according to the manufacturer's instructions to glue the gels to the bottom of a multi-well plate. Place a small droplet of Polymethyl soane at the center of each well using forceps. Place one poly acrylamide gel in each well flexible plastic support side down.

After curing the poly dimethyl suboxane, place a small amount of the previously prepared cellulose sampa in each well with a transfer pipette and swirl from side to side to coat the gel surface evenly. Place the well plate under a high intensity UV lamp for five minutes. When finished, rinse the gels with PBS to remove excess cello.

Following this pipette, approximately 50 microliters of the previously prepared collagen Type one solution into each. Well leave the plate covered at room temperature for at least two hours after rinsing with PBS to remove excess collagen solution. Sterilize the gels under UV light in a tissue culture hood for two hours.

Finally soak the gels in complete medium overnight to hydrate and equilibrate. Use gels for cell seating immediately or store in the refrigerator for up to two days young's modulus as a function of several acrylamide and bis. Acrylamide concentrations designated as A and B respectively is shown here as anticipated.

Both storage modulus G prime and loss modulus G double prime are independent of frequency. It was also confirmed that drying and then rehydrating the gels did not affect their young's modulus. By using the Crosslinker Sulo sampa, a uniform collagen coating on hydrogels of any stiffness can be achieved.

It was observed that when seeded on poly acrylamide gels for 24 hours, breast cancer M-D-A-M-B 2 31 cells remained round on the soft 0.5 and one kilo pascal gels, but were able to spread and elongate on the stiff 100 kilo pascal gel. In addition, hydrogels made on top of the flexible plastic support are transparent and allow for easy visualization and microscopy. Furthermore, the flexible plastic support does not auto fluoresce and thus does not interfere with the imaging of fluorescently labeled cells.

Cell morphology was further quantified in terms of overall cell spreading area and cell circularity. After watching this video, you should have a good understanding of how efficiently and inexpensively to assemble polyamide gels in a multi-well plate format. For the study of stiffness dependent cell biology.