3.9 Data
Interpretation
1. For each experimental condition, compute the difference
between the tension sensor and tension-less control FRET
efficiencies (see Note 20).
2. Use previously published FRET efficiency to force calibration
[2] to retrieve the force.
3. Mind MTM limitations in your interpretation [1].
4 Notes
1. There exist other strategies for tension-less constructs, which
may or may not give equivalent results depending on the
protein of interest, as discussed in [1].
2. High magnification is irrelevant but comes with high numerical
aperture, which increases resolution and signal intensity, and
should also come with apochromatic correction. Choose the
highest you can, typically 1.4 for standard objectives.
3. Save used collagen solution back in the final concentration
solution for later use. Store at 4
C indefinitely.
4. Use the UV light of the cell culture hood. Do not cover the
dishes with a plastic lid. Multiple dishes can be prepared in
advance and stored on the shelf indefinitely.
5. When re-suspending cells, pipette up and down to shear and
dissociate cell aggregates. This will facilitate homogenous
seeding.
6. When seeding, make sure the cell suspension covers the whole
coverslip by gently shaking the dish linearly in X and Y, not
circularly as cells would aggregate at the center of the coverslip.
7. Execute with care and promptly directly on the microscope
stage to keep focus, temperature and position the same.
8. Make sure there is enough oil to cover the whole surface of the
imaging medium to prevent evaporation.
9. Check that the monolayer is confluent before this step.
10. Two perpendicular scratches maximize wound edge length per
coverslip. Use/draw fiducial markers on the coverslip/imaging
chamber to easily find the wound once on the microscope
stage.
11. Selecting positions in close proximity minimizes the spreading
and loss of the objective immersion medium.
12. Consider the total acquisition time per time-point (multiple
positions and Z sections), the total duration of the time-lapse,
photobleaching and phototoxicity. 20- to 30-min intervals are
adequate for most experiments.
Molecular Tension Microscopy of E-Cadherin During EMT
297
Interpretation
1. For each experimental condition, compute the difference
between the tension sensor and tension-less control FRET
efficiencies (see Note 20).
2. Use previously published FRET efficiency to force calibration
[2] to retrieve the force.
3. Mind MTM limitations in your interpretation [1].
4 Notes
1. There exist other strategies for tension-less constructs, which
may or may not give equivalent results depending on the
protein of interest, as discussed in [1].
2. High magnification is irrelevant but comes with high numerical
aperture, which increases resolution and signal intensity, and
should also come with apochromatic correction. Choose the
highest you can, typically 1.4 for standard objectives.
3. Save used collagen solution back in the final concentration
solution for later use. Store at 4
C indefinitely.
4. Use the UV light of the cell culture hood. Do not cover the
dishes with a plastic lid. Multiple dishes can be prepared in
advance and stored on the shelf indefinitely.
5. When re-suspending cells, pipette up and down to shear and
dissociate cell aggregates. This will facilitate homogenous
seeding.
6. When seeding, make sure the cell suspension covers the whole
coverslip by gently shaking the dish linearly in X and Y, not
circularly as cells would aggregate at the center of the coverslip.
7. Execute with care and promptly directly on the microscope
stage to keep focus, temperature and position the same.
8. Make sure there is enough oil to cover the whole surface of the
imaging medium to prevent evaporation.
9. Check that the monolayer is confluent before this step.
10. Two perpendicular scratches maximize wound edge length per
coverslip. Use/draw fiducial markers on the coverslip/imaging
chamber to easily find the wound once on the microscope
stage.
11. Selecting positions in close proximity minimizes the spreading
and loss of the objective immersion medium.
12. Consider the total acquisition time per time-point (multiple
positions and Z sections), the total duration of the time-lapse,
photobleaching and phototoxicity. 20- to 30-min intervals are
adequate for most experiments.
Molecular Tension Microscopy of E-Cadherin During EMT
297