The overall goal of this procedure is to prepare DNA cross-linked poly acrylamide hydrogels that can stiffen or soften by the pipetting of single stranded DNAs into the culture media after cells are plated on top. This is accomplished by first Resus suspending Lyophilized SA one, SA two L two R two, and control single-stranded DNAs in buffer and individually polymerizing the SA one and SA two solutions into a poly acrylamide acrylamide backbone. The second step is to mix the SA one and SA two polymerized solutions with the L two solution to form the DNA gel.
Next, the DNA gel solution is then immobilized onto glass cover slips with optical glue. The final step is functionalizing the gels by treating them with sul sampa and then PDL. Ultimately, cells are plated on the DNA gels and gel elasticity is modulated with the addition of R two L two or control single stranded DNA to soften stiffen or not change gel elasticity respectively.
Results from these experiments show that the dynamic nature of the underlying substrate direct cell behavior, The advantage of using this technique over other methods such as CROs, lingo, ball hydrogels, is that DNA gels do not require a stimulus such as UV to modulate their elasticity. Instead, DNA gels are stiffened and softened via the diffusion of L two or R two, single-stranded DNA through the gel respectively. First centrifuge lyophilized SA one single stranded DNA at 2000 GS for 15 seconds to ensure that all the single stranded DNA is at the bottom of the tube.
After centrifugation, prepare three millimolar of SA one solution by adding 107 microliters of one x tris, EDTA or TE buffer to the tube Heat the SA one NTE buffer at 70 degrees Celsius for five minutes or until the single stranded DNA pellet is completely dissolved. Following this, prepare the SA one polymerized solution by adding 45 microliters of 40%acrylamide and 18 microliters of 10 x tribo, EDTA or TBE buffer and centrifuge. This mixture to facilitate mixing.
Insert a P 200 tip, attach to a nitrogen gas source into the solution and slowly allow nitrogen gas to pass through the solution for two to three minutes. Next, centrifuge the sample for 15 seconds at 2000 Gs to gather the solution When finished at 4.5 microliters of 2%ammonium per sulfate or a PS to the sample to initiate gel polymerization, invert the tube several times to mix After centrifuging the sample to gather the solution for homogenous polymerization, add 4.5 microliters of 20%tetraethyl methylene, diamine, or teed to the sample to catalyze the polymerization Once the sample has undergone, mixing and centrifugation. Degas the solution with nitrogen gas for three to five minutes to complete polymerization and minimize unreactive monomers.
Following this, incubate the solution for 10 minutes at room temperature to complete polymerization and afford the SA one polymerized solution. Repeat the previous steps with lyophilized SA two single-stranded DNA adding 45 18 4 0.5 and 4.5 microliters of acrylamide T-B-E-A-P-S and teed respectively to 108 microliters of the SA two solution. To prepare the L two R two and control solutions, centrifuge, lyophilized, single stranded DNA and add the following amounts of TE buffer to the corresponding lyophilized single-stranded DNA as previously described, heat the solution until dissolved for the gel solution.
Preparations heat the s sa one and SA two polymerized solutions at 70 degrees Celsius for approximately one minute. Until solution viscosity is reduced. Add 10 microliters or 10 parts of the SA one polymerized solution to 10 microliters or 10 parts of the SA two polymerized solution.
Then mix the SA one and SA two polymerized solutions together via alternating heating for 15 seconds at 70 degrees Celsius and stirring for 15 seconds with a pipette tip for a total of one minute. After mixing at six microliters or six parts of 100%L two solution to form a 100%gel. Once the sample has been mixed as previously described, pipette the 100%gel into a 60 millimeter Petri dish and heat the gel for one hour at the optimal annealing temperature.
Next, allow DNA crosslinks to continue to re hybridize by incubating the gels for four hours at room temperature. Cover the gel with PBS containing calcium at magnesium following incubation overnight at room temperature, remove the remaining buffer from the Petri dish and place the gel into a micro centrifuge tube. At this point, heat the 100%gel at 70 degrees Celsius for about 30 seconds or until the gel is less viscous.
For pipetting place glass cover slips onto a 70 degree Celsius heat block and allow them to heat for one to two minutes. Add a drop of optical adhesive to each glass cover slip. Then add 20 microliters of the 100%gel to each glass cover slip, and allow the gel to melt and spread over them.
Remove each glass containing gel from the heat block and place them into a 24 well tissue culture dish. Expose the gels to a 365 nanometer UV light for 15 minutes. After heating the gel for one hour at the optimal anal temperature and allowing the DNA crosslinks to re hybridize for four hours, add PBS to them.
Then incubate the gels overnight at four degrees Celsius. Following incubation, remove the buffer from the wells with a pipette being careful not to aspirate the gel. Then add sulfa sampa to cover each gel.
Once the gels have been exposed to 365 nanometer UV light for 15 minutes, remove the sulfa sampa. Rinse the gels once with PBS to remove the excess sulfa sampa after repeating the previous steps, incubate the gels in approximately 300 microliters of 0.2 milligrams per milliliter of poly de lycine overnight at four degrees Celsius When finished, wash the gels twice with PBS for five minutes to remove excess polylysine and add media. Once the gels have been incubated in media for 30 minutes, remove the media immediately before plating the cells for cell culturing and imaging.
Fibroblasts grown on stiffening gels, had no alterations in aspect ratio, but exhibited a larger projection area than fibroblasts grown on 100%gels. Fibroblasts grown on 100%Gels had a smaller projection area and larger aspect ratio than fibroblasts grown on 80%gels. These results indicate that fibroblast behavior is dependent on the dynamic nature of the microenvironment.
Neurons grown on softening gels had no differences in primary dendrite number or axon length, but exhibited shorter primary dendrites than neurons grown on 50%gels. As in fibroblast studies, neuro phenotypes on static gels were dissimilar to neuro phenotypes on dynamic gels. Neurons grown on 50%gels had fewer primary dendrites and longer axons than neurons grown on 100%gels.
While attempting this procedure is extremely important to remember to accurately pipette the viscous gel, since concentrations are important in avoiding gel bursting, accurate pipetting may take several attempts, and we offer several other options in the discussion portion of this protocol.
