In this method, the force is directly applied to the nucleus. This decouples the force transmission effect from the cell plasma membrane and cytoskeleton, revealing the molecular mechanisms of nuclear mechanosensing. The force is applied within the living cells in a non-invasive way.
Compared to optical tweezers, the magnetic field and magnetic force do not affect the cell functions and have higher throughput. Begin culturing the cells with magnetic microbeads by weighing 0.2 grams of carbonyl iron microbeads having seven micrometer of mean diameter. Suspend the microbeads in one milliliter RPMI 1640 culture medium using a pipette.
Then take the Petri dish with B2B cells to the biosafety cabinet and quickly add 200 microliters of the medium containing microbeads into the Petri dish. Put the Petri dish in an incubator until microbeads are internalized by the cells. Open the inverted microscope and using confocal fluorescence imaging, determine the optimal time for the internalization of the cell lines every six hours by visualizing the microbead, nuclear, and cell boundary.
The internalized microbeads will be present within the cell boundary. For small force application and live-cell imaging, open the software application Elements. To define the configuration magnetic find, click the one over two button to set the scanning speed to one frame per two seconds.
Set pinhole size to 1.2 Airy unit. Check only the FITC channel and set PMT HV as 70, offset as zero, laser intensity as 10. To define the configuration magnetic YAP nucleus, set the scanning speed to one frame per four seconds by clicking the one over four button.
Then click the 1.2 Airy unit button to set the pinhole size to 1.2 Airy unit and check the FITC channel. Set PMT HV as 70, offset as zero, laser intensity as 10. For imaging the nucleus boundary and nuclear stain intensity, check the cyanine 5 channel.
Do not click the 1.2 Airy unit button and set PMT HV to 70, offset to zero and laser intensity to 10. The pinhole size will be optimized for three-dimensional YES-associated protein imaging. Next, turn on the DIA through Elements.
Open spin view, use a brightfield and adjust the focus of the object to get a clear in-focus image of cells. Use a 10X objective to find appropriate multiple single cells in three conditions, such as a cell with a single microbead inside, with multiple microbeads inside, and without any microbead inside. Then switch to 40X objective and name this position with the appropriate position number.
Open Elements, click on magnetic find, remove interlock, then scan tabs, then select the top and bottom buttons to adjust the Z position of the focal plane for setting the lower and upper limit for the Z stack of the selected cells. Stop scanning by clicking scan again. Switch to magnetic YAP nucleus configuration and set the filename as before_small_force.nd2.
Click on the run button with the recorded Z stack. Switch to the right light path and turn on DIA. Open spin view and click on the recording button.
Move the magnet to 46 millimeters above the Petri dish bottom by spinning the knob of the magnet moving device. Save and check the video to confirm the microbeads displacement induced by magnetic force. Repeat the scanning procedure by setting the filename as after_small_force.nd2.
Next, switch to the right light path, turn on DIA and open spin view before clicking on the recording button. Spin the magnet moving device knob up to 120 millimeters above the Petri dish bottom and save a brightfield image sequence or video. Repeat the steps for scanning by setting the filename to before_large_force.nd2.
The microbeads cannot emit fluorescence under laser excitation in the FITC or cyanine 5 channel. Thus, the internalized microbeads were identified by the dark hollow located in the confocal imaging of fluorescence of YES-associated protein and nucleus. Nuclear circularity showed no significant difference between control cells and cells with microbead internalization.
The YES-associated protein cell nucleus-to-cytoplasm ratio of control cells co-cultured with microbead but without internalization and cells with microbead internalization also showed no significant difference. The nucleus deformation and de-polymerization of actin were caused by the compression force applied by the microbeads in cytoskeleton-containing cells. The quantitative imaging analysis calibration curve provided by the quantitative AFM force displacement relationship.
Based on this relationship, the beads applied force was estimated. The nucleus deformation and YES-associated protein translocation were observed on application or the release of the magnetic force. The intensity changes in YES-associated protein in the green channel and no change in the nucleus staining in the red channel confirmed that the magnetic force-induced nuclear deformation triggered by the YES-associated protein translocation.
The quantification of the net change of the YES-associated protein nucleus-to-cytoplasm ratio within two groups confirmed that the magnetic force applied to the microbeads within the cytoplasm induced the YES-associated protein translocation and changed the YES-associated protein nucleus-to-cytoplasm ratio. Before applying force and imaging, ensure that the cells have internalized the magnetic microbeads and the beads are within the cell boundary.
