This protocol offers a method to map the molecular forces generated by cells and particularly immune cells. The protocol is critical to labs interested in studying mechanotransduction in mechanobiology. The main advantage of this technique is that it is super easy and allows one to use conventional fluorescence microscopy to quantify force transmission in immortalized and primary cells.
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%aminopropyltriethoxysilane for one hour at room temperature to initiate the reaction with the hydroxyl group on the coverslips.
Rinse the surface of the coverslips 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 a 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 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 one 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 one 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 ten to one 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 four 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 one 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 one 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. A successful surface had a pale pink color.
The high quality of the surface was demonstrated with a clean background and the reflection interference contrast microscopy or RICM channel and uniform fluorescence intensity in the Cy3B channel. Time-lapse images of tension locking indicated that both TCR anti-CD3 epsilon and TCR-peptide-MHC tension signals were amplified because of hairpin locking and signal accumulation. The locking strand recorded short-lived mechanical pulling events of TCR peptide MHCN4 starting at zero minutes, which facilitated quantitative analysis and revealed the distinct pattern of the TCR peptide MHC force that resembled the bullseye pattern of immune synapses.
Every step, your intention probe substrate preparation needs attention to detail and careful handling of the materials. This technology enables visualization of transient molecular forces that occur at the cell surface when it interacts with the environment, especially in the immune cells in search for antigens.
