To model in vivo tissue compliance using acrylamide hydrogels. This is accomplished by first generating reactive bottom cover slips and siliconized top cover slips. The second step of the procedure is to pour and create the acrylamide hydrogels.
The third step of the procedure is cross-linking the extracellular matrix protein of choice to the hydrogel. The final step of the procedure is incubation and analysis of cells. Ultimately, results can be obtained that show how varying extracellular matrix compliance in vitro regulates cell behavior through analysis using immunofluorescence microscopy, real-time quantitative PCR and western blotting.
Today we'll show you the procedure for the generation and use of acrylamide hydrogels. We use this procedure in our laboratory to study our extracellular matrix stiffness regulate cell morphology, cell signaling and proliferation. So let's get started.
Begin this procedure by generating reactive cover slips. First place a layer of paraform on the bottom half of a 150 millimeter Petri dish. Then transfer up to nine autoclave 25 millimeter cover slips onto the para film and cover them with one milliliter of 0.1 molar sodium hydroxide.
Incubate the cover slips for three minutes Following incubation, aspirate the sodium hydroxide with a vacuum line working in a chemical hood pipette 0.5 milliliters of three amino profile trimethyl or three A-P-T-M-S on each cover slip. Incubate the cover slips for three minutes and then aspirate the three A-P-T-M-S. Take caution not to incubate too long to avoid the formation of foam on the cover.
Slips following treatment. Rinse the cover slips once with 20 milliliters of deionized water in the same dish. Remove the cover slips from the dish using curved forceps and transfer them treated side facing up to a new 150 millimeter dish.
Then wash the cover slips with deionized water again and place them on the rocker for 10 minutes. Following incubation, remove the water and wash an additional two times. It is very important to remove all of the three A-P-T-M-S so that it will not react with the glutaraldehyde and leave a cloudy white precipitate 10 minutes prior to use for the glutaraldehyde.
Using curved forceps, transfer the cover slips to a clean dish layered with param and aspirate any remaining liquid Using a vacuum line, then cover each cover slip completely with 0.5 milliliters of 0.5%glutaraldehyde in sterile deionized water to cross-link the three A-P-T-M-S and the poly acrylamide gel. Incubate the cover slips for 30 minutes in a chemical hood. Then aspirate the glutaraldehyde rinse and wash the cover slips again with deionized water.
Then dry the cover slips completely. Add new cover slips to a 50 milliliter Falcon tube containing a 10%surface seal solution in chloroform and rock for at least 10 minutes. Decant the surface seal solution and air dry.
The cover slips on Kim wipes in the biological safety cabinet where the hydrogels will be prepared to begin hydrogel preparation. Use curved forceps to transfer the cover slips reactive side up to a sheet of paraform that has been taped onto the surface of the biological safety cabinet. Be sure that the cover slips are flat on the paraform surface.
Then prepare a saturated and hydroxy CIN aide or NHS solution in toluene by dissolving a small amount of NHS in enough toluene. For the specific experiments, add small amounts of NHS until the NHS no longer dissolves. The saturated solution is usually cloudy and pink.
Next, prepare the acrylamide BIS acrylamide water and a PS to reach the desired acrylamide percentage. Add the reagents to the micro use tubes as outlined in the written protocol. Then one aliquot at a time.
Add the NHS and TM me. Vortex the tube briefly and immediately Pour between three to five gels using 140 microliters per cover slip in the biological safety cabinets. Quickly place the SILICONIZED 25 millimeter cover slip on top of each gel before it begins to polymerize.
Addition of the top cover slip will allow the acrylamide to completely cover the bottom cover slip. Incubate this sandwich at room temperature until the acrylamide polymerizes to determine when polymerization has occurred. Check the residual acrylamide solution in the micro centrifuge tube.
The polymerization will take a few minutes for stiff gels and a bit longer for soft gels. Once polymerization has occurred carefully pick up the sandwich wearing sterile gloves. Then slide off the top cover slip until it overhangs the polymerized gel.
Then pry the cover. Slip off the gel, discard the top cover slip. Place each bottom gel cover slip hereafter called the hydrogel into a six well plate.
Next, add two milliliters of phosphate buffered saline or PBS per well of a six well plate. Then wash the hydrogels with PBS and incubate on a rocker for five minutes. Repeat this wash two times.
To begin the experiment, cover each hydrogel with two milliliters of a fibronectin solution or other extracellular matrix protein. Incubate the protein on the hydrogel overnight at four degrees Celsius to allow it to covalently bind to the hydrogel following overnight incubation aspirate the ECM solution. Then block the unreactive NHS with one milligram per milliliter of heating activated fatty acid-free bovine serum albumin in serum free media and incubate for at least 30 minutes at 37 degrees Celsius.
Following incubation, wash the hydrogels once with sterile PBS. Next plate the cells in the appropriate cultured medium containing fetal bovine serum. Onto the hydrogel here.
Establish mouse embryonic fibroblasts are used. Determine the number of cells to be seeded on the hydrogel based on the degree of cells spreading and confluence required for experimentation. Approximately 10 to the five cells necessary for western blot and quantitative PCR analysis.
Then incubate the cells under the appropriate conditions for the specific cell type. Following the incubation period. Extract the cell protein or mRNA according to need.
Pipette 100 microliter droplets of lysis buffer onto a sheet of paraform on the lab bench, leaving approximately two to three centimeters between each droplets. Next, carefully remove each hydrogel by lifting the bottom cover slip from the well. Using curved forceps and placing its cell side down on top of the droplets.
Incubate the cells with the lysis buffer for exactly one minute. Finally, remove the cover slips and transfer the lysis buffer to a micro centrifuge tube. Alternatively, when extracting RNA transfer each hydrogel to a new six well plate and add one milliliter of triazole per well.
Incubate the gels for three minutes following incubation. Remove the triazole solution for storage in a micro centrifuge tube. Thorough washing of the cover slips following addition of A-P-T-M-S is an important step in producing reactive cover slips.
Properly washed and dry cover slips are free of any precipitate residual. A-P-T-M-S will react with the glutaraldehyde and produce a white cloudy precipitate. If the precipitate, the whole procedure must be repeated as the cover slip is no longer usable.
Following hydrogel formation and coating with ECM proteins, overnight cells are seeded. There is a distinct difference between cells spreading on stiff versus soft hydrogels as can be seen by foid in staining and mouse embryonic fibroblasts cells spread to a greater extent on stiff as compares to soft hydrogels. Indeed, most cells attaching to a soft hydrogel will remain compact and attach less efficiently.
Representative quantitative PCR results of cycling D one mRNA levels in mouse embryonic fibroblasts show that cycling D one is significantly upregulated on a stiff matrix, but not on a soft matrix. This suggests that matrix stiffness regulates cell cycle progression in vitro. We've just shown you how to generate hydrogel severing stiffness, modeling in vivo physiological conditions When doing this procedure is important to remember to make extra reactive cover slips as accident and error will occur.
So that's it. Thanks for watching and good luck with your experiments.
