Using DNA-Based Tension Probes to Assess Receptor Forces Applied by Immune Cells
Trascrizione
To begin, place 25-millimeter coverslips on a polytetrafluoroethylene rack in a 50-milliliter beaker, and rinse the coverslips three times by submerging them in water. Mix equal volumes of ethanol and water, and add 40 milliliters of this solution to the beaker containing the rack and the coverslips. Then, seal the beaker using parafilm tape.
Clean the coverslips by sonicating the beaker for 15 minutes in an ultrasonic cleaner, and discard the liquid after sonication. Then, rinse the beaker containing the rack and coverslips with water, at least, six times to remove any remaining organic solvent. To prepare a fresh piranha solution, add 30 milliliters of sulfuric acid to a 50-milliliter beaker and slowly add 10 milliliters of hydrogen peroxide. Gently mix the piranha solution using the end of a glass pipette.
To initiate etching, transfer the rack holding the coverslips to the beaker containing piranha solution, and incubate for 30 minutes at room temperature to cleanse and hydroxylate the coverslips. Then, transfer the rack using steel or polytetrafluoroethylene tweezers to a clean 50-milliliter beaker and rinse with water six times. In order to remove water completely, immerse the rack holding the coverslips in a 50-milliliter beaker with 40 milliliters of ethanol, then, discard the ethanol.
Next, immerse the rack in 40 milliliters of ethanol containing 3% aminopropyl triethoxy silane for 1 hour at room temperature to initiate the reaction with the hydroxyl group on the coverslips. Rinse the surface of the coverslip six times by submerging them in 40 milliliters of ethanol, and dry them in an oven at 80 degrees Celsius for 20 minutes.
Cover the inner bottom of the Petri dish with parafilm tape to prevent the coverslips from sliding inside the Petri dish. Then, place the amine-covered coverslips with the side to be functionalized with DNA tension probes facing up. Cover the coverslips at minus 20 degrees Celsius for future use.
To modify the amine groups on the coverslips, add lipoic acid-PEG-NHS and mPEG-NHS and incubate the Petri dish at room temperature for 1 hour. At the end of the incubation, rinse the surface three times with water. After that, add 100 microliters of 0.1 molar sodium bicarbonate containing 1 milligram per milliliter of Sulfo-NHS-acetate to a set of sandwich coverslips, and incubate for 30 minutes for passivation to occur.
Add 0.5 milliliters of gold nanoparticles to each coverslip and incubate for 30 minutes at room temperature. After incubation, rinse the coverslips with water three times. After adding black hole quencher 2 strand to the annealed DNA tension probe at a 10-to-1 ratio, add 100 microliters of the mixture per two coverslips to make the sandwich.
Carefully place a wet lab tissue ball in the Petri dish away from the coverslips, and seal the dish with parafilm tape to prevent the solution from drying. After that, cover the dish with a foil, and incubate it at 4 degrees Celsius overnight. On the next day, assemble the imaging chamber taking care to prevent surface cracking while tightening the chamber. Then, add 0.5 to 1 milliliters of Hank's balanced salt solution to the imaging chamber.
Use the Cy3B channel with a 100x objective to capture fluorescent signals of cells plated onto the DNA hairpin tension probes when they start to spread. After preparing 100 micromolar non-fluorescent locking strand stock, at the desired time point, add it to the cells at a final concentration of 1 micromolar in the imaging chamber to store the tension signal, and mix it with the cells by pipetting.
After about 10 minutes of locking, acquire time-lapse movies or endpoint images in epifluorescence for both qualitative tension mapping and quantitative analysis.
