The overall goal of this experiment is to explore the biochemical pathways that are activated in response to tension using ligand coated magnetic microbeads and forced application to adhesion receptors. This method can help answer questions in the mechanic transaction field such how mechanical tension from the express helometrics directs them from differentiation or how tissue compliance drives their transformation. The main advantage of this technique is that many cells can be stimulated for chemical analysis by using beads to apply tension on specific subset of adhesion receptors.
To begin, thoroughly resuspend the super paramagnetic tosylactivated beads in the original vial by vortexing for at least 30 seconds. Aliquot 40 microliters of the resuspended superparamagnetic beads to a 1.5 milliliter microcentrifuge tube containing one milliliter 0.1 molar sodium phosphate pH 7.4. Next, place the tube on a magnetic separation stand to separate the magnetic beads from the solution.
Discard the supernatant and remove the tube from the magnetic separation stand. Resuspend the beads in one milliliter 0.1 molar sodium phosphate pH 7.4. After washing the beads twice, resuspend the beads in one milliliter of the same buffer.
Then, combine 100 micrograms of bovine fibronectin or any other cell adhesion receptor ligand to the beads and vortex. Repeat this step by replacing the ligand with BSA, poly-D-lysine, or apotransferrin for negative controls. Incubate the beads with the ligand containing solution for 12 to 24 hours at 37 degrees Celsius on a rotor.
If bead aggregates appear after the reaction has occurred, sonicate for no more than 10 to 20 seconds. Next, isolate the beads using a magnetic separation stand and aspirate the remaining solution. Then add one milliliter of a PBS solution supplemented with 0.2%bovine serum albumen.
Incubate for one hour on a rotor at 37 degrees Celsius. Using a magnet, wash the beads with one milliliter of PBS solution supplemented with 0.2%bovine serum albumen two times. Before resuspending them in one milliliter of the same buffer.
Sonicate the beads if aggregates appear for no more than 20 to 30 seconds. Proceed to the cell assays, or store the beads at four degrees Celsius for up to one month. Culture adherent cells on a 60 milliliter tissue culture dish in appropriate growth medium until reaching 80%confluency.
After pelleting the ligand coated beads, Resuspend the pellet in one milliliter of PBS solution supplemented with 0.2%bovine serum albumen. Add 100 microliters of the ligand coated bead solution to five milliliters of warm culture medium and vortex. Next, aspirate the medium in a 60 milliliter culture dish and add five milliliters of warm growth medium supplemented with beads.
Incubate the suspension for 20 minutes under cell culture conditions to allow the beads to sediment and adhere to the cells. It is crucial not to exceed 20 minutes, since beads may be internalized by phagocytosis. Next, observe the beads under a light microscope using a 10X or 20X objective.
Check for bead adhesion by slightly shaking the dish to discriminate between attached and floating beads. Place a round 38 millimeter neodymium magnet onto the upper face of a normal 60 millimeter culture dish lid and keep it held in place using two smaller 13 millimeter neodymium magnets positioned on the lower face of the lid. Incubate the cells subjected to tension for the desired time points and cell culture conditions.
Swap the normal dish lid for a lid with the magnet. Since the magnet are very powerful, manipulate them carefully. After treatment, place the dish on ice and carefully aspirate the entire medium from the dish.
Add 300 microliters of cell lysis buffer and incubate for 10 minutes on ice. Then, collect the lysate using a cell scraper and transfer it to a pre-chilled 1.5 milliliter microcentrifuge tube. Pellet the magnetic beads using the magnetic separation stand and transfer the total cell lysate to a new, pre-chilled 1.5 milliliter microcentrifuge tube.
Remove a 15 microliter aliquot from the total cell lysate obtained after bead separation for western blot analysis. Wash the beads three times with one milliliter of ice cold lysis buffer. Then, add 50 microliters sample buffer to the bead pellet.
After mixing the suspension by vortexing, boil it at 95 degrees Celsius for five minutes using a dry block heater. This fraction contains the isolated adhesion complexes. Proceed to biochemical analysis or store the fraction at minus 20 degrees Celsius.
This figure is shown to demonstrate the purification of the adhesion complexes. GAPDH immunoblotting is used as a loading control for total cell lysate and to check the purity of the adhesion complexes. A long exposure was used to validate the absence of cytoplasmic signal in the purified adhesion complex fraction.
Fibronectin coated beads were used to investigate the mechano-transduction processes that occur over time at the adhesion complexes in response to tension. After magnetic separation of the adhesion complex fraction, the lysate and the adhesion complex fraction were analyzed by western blot. Loading control and candidates known to be recruited or phosphorylated at adhesion complexes in response to mechanical tension were immunoblotted.
While tension did not affect Paxillin recruitment to the adhesion complexes, its phosphorylation on tyrosine 31 was enhanced in response to mechanical tension both in the total cell lysate and in the adhesion complex fraction. After watching this video you should have a good understanding of how to apply tensional forces to adhesion surface proteins using magnetic beads. Once mastered, this technique can be done in two hours if it is performed properly.
While attempting this procedure it's important to remember to be cautious with the powerful magnets. Following this procedure biochemical studies such as western blots, actin polymerization assay or enzymatic assay can be performed to answer additional questions like whether kinase activity is regulated by tension. This technique may help researchers in the field of cell biology to explore magnetic sensitive signaling pathways in cells.
