This protocol is significant due to the simplicity of the TGT sensor platform used to study EGFR-integrin crosstalk and its influence on cell mechanical forces. Rao outlined to investigate EGF-dependent regulation of cell mechanics. The protocol is adaptable for different ligands, cell types, stimulation paradigms, and can be coupled with TGTs of varying tension thresholds.
Surface synthesis may seem intimidating at first. The key is to be prepared and organized to precisely execute the intricate steps by employing a checklist for reagent preparation and calculations. On day one, place up to eight 25-millimeter glass cover slips into a polytetrafluoroethylene rack.
Place the rack in a 50-milliliter borosilicate beaker containing 40 milliliters of 200-proof ethanol. Sonicate at an operating frequency of 35 kilohertz for 10 to 15 minutes at room temperature. Fill a 50-milliliter beaker with 40 milliliters of piranha solution freshly prepared by mixing sulfuric acid and hydrogen peroxide in a 3:1 ratio in a Pyrex beaker and stirring with a glass pipette.
Transfer the cover slip rack into the beaker and incubate for 30 minutes at room temperature in the fume hood to etch the cover slip surface. After etching, transfer the cover slip rack to a beaker with ultrapure water with tweezers. Visually inspect the cover slips to ensure the surfaces look clean with no patterns or dust particles on the glass surface.
Transfer the cover slip rack to a beaker with 200-proof ethanol and wash twice for 15 seconds to equilibrate surfaces to organic solvent. Transfer the cover slip rack into 200-proof ethanol solution with 3%APTES for one hour at room temperature to silanize the cover slips. Immerse the rack in a clean beaker with 200-proof ethanol solution.
Dry the cover slips using nitrogen gas with low exit pressure. Place the cover slips into a 10-centimeter polystyrene dish with a piece of parrafin film laid flat inside it. Add 100 microliters of two milligrams per milliliter NHS-biotin solution in DMSO to four cover slips placed on paraffin film.
Set a sandwich with the other four cover slips on top, with two cover slips facing toward each other with the functionalization solution in between. On day two, remove the dish from four degrees Celsius and separate the sandwiched cover slips. Avoid scratching or damaging the functionalized surface while separating the sandwich.
Then, orient the cover slips in the rack with the coated surface facing each other. Dry with nitrogen gas. Place the cover slips in a new dish with a parrafin film inside it.
Add 800 microliters of 0.1%bovine serum albumin in 1X PBS to each cover slip. Incubate the cover slips at room temperature for 30 minutes to passivize the surface and block nonspecific binding of subsequent functionalization reagents. To functionalize the cover slips, add 800 microliters of one microgram per milliliter of streptavidin in 1X PBS at room temperature for 45 to 60 minutes.
Assemble the TGT probes in a PCR tube using a thermocycler. After incubation, wash the cover slips three times with 1X PBS. Add 100 microliters of the preassembled TGT probes to four cover slips and make sandwiches using the remaining four cover slips with the functionalized side facing the probes.
Cover with aluminum foil. After incubation, separate the sandwiches and wash the cover slips with 1X PBS three times. Carefully assemble the cover slips into pre-cleaned imaging chambers.
To investigate the effect of the epidermal growth factor stimulation on Cos-7 mechanics, adhesion, and cell spreading, trypsinize Cos-7 cells with 0.05%trypsin-EDTA for two minutes. Neutralize the trypsin by washing with HBSS and centrifuging at 800 G for five minutes. Plate cells at a density of 40, 000 cells on the assembled TGT surfaces in DMEM supplemented with 50 nanograms per milliliter EGF or DMEM without EGF.
Following incubation, wash the cells three times with 1X PBS. Fix with two milliliters of 4%paraformaldehyde for 12 minutes at room temperature. Wash the cover slips five times with 1X PBS at five-minute intervals at room temperature.
Optionally, incubate the cover slips with 50-millimolar ammonium chloride in 1X PBS for 30 minutes at 37 degrees Celsius. Add buffer A and place the imaging chambers with cover slips in a humidity container. Incubate for 30 minutes at 37 degrees Celsius to block and permeabilize the cells.
Dilute the primary anti-paxillin antibody at 1:250 dilutions in blocking buffer. Incubate with 200 microliters of primary antibody solution per cover slip for two hours at 37 degrees Celsius. Label cells simultaneously with a mixture of di-conjugated goat/anti-rabbit secondary antibody at 1:800 dilution and di-conjugated phalloidin at 1:400 dilutions in 200 microliters of blocking buffer per cover slip.
Wash the surfaces five times with 1X PBS at five-minute intervals. To begin, add oil to the objective, clean the cover slip bottom of the sample chamber, and place the sample on the stage. Focus on a cell and engage the perfect focus.
Align the RICM by closing and centering the epi-illumination aperture diaphragm. Focus the 488-nanometer laser to a small spot on the ceiling of the room and increase the angle of incidence until past the critical angle, while monitoring fluorescence on the camera in a live mode. Observe a sharp reduction in background fluorescence and a single in-focus plane when the critical angle is surpassed.
Identify cells for imaging using the live mode of the camera using RICM. Acquire the RICM and TIRF images of actin at 488-nanometer excitation, integrin tension at 561-nanometer excitation, and paxillin at 647-nanometer excitation. Obtain images sequentially using an exposure time of 200 milliseconds.
Change cover slips, focus, and repeat. Cos-7 cells were plated on this TGT surface with or without EGF stimulation to study the impact of EGFR activation with ligand stimulation on integrin mechanics. If an integrin binds the ligand and applies a force larger than the tension threshold total of the probe, the DNA duplex will separate, leading to fluorescence.
Any TGT probe that a mechanical force has not ruptured will remain non-fluorescent. The cells were incubated with or without EGF on the TGT surfaces for 60 minutes, fixed, and immunostained to display the focal adhesion distribution and the organization of the cytoskeleton. In the RICM image, Cos-7 cell spreading on the 56-piconewton TGT surface was significantly enhanced with EGF stimulation compared to without stimulation.
Stimulation with EGF resulted in a more circular morphology, representing Cos-7 cell spreading and growing. The fluorescence from open probes is also higher with EGF stimulation, as observed in the tension fluorescence image. The integrated intensity of open probes proportional to the number of open probes was much higher with EGF stimulation than without stimulation.
Always remember the orientation of the cover slips, keeping the functionalized surface facing upwards. When separating the sandwich, be gentle so the surface is not scratched or damaged. Following surface synthesis, one can probe for cytoplasmic proteins regulating cell adhesion or mechanic transduction.
This allows identification of downstream signaling molecules involved in orchestrating cell mechanics at the plasma membrane.
