3 Methods
Carry out all procedures in a cell culture hood unless otherwise
specified.
3.1 Cell Culture
Maintenance
Pass MDCK II cells three times a week as follows:
1. Prepare a new cell culture flask containing 3 mL of fresh culture
medium and put it into the incubator.
2. Remove old culture medium and dead cells from the cell culture flask.
3. Wash once with 1 mL of washing medium and by gently
shaking the cell culture flask.
4. Add 1 mL of Trypsin-EDTA solution in DPBS to the cell
culture flask.
5. Put the cell culture flask into incubator and wait about 10 min
for cells to detach from the surface and from each other.
6. Use a microscope to evaluate cell detachment.
7. Once cells are detached, transfer 1:10 of the collected volume
into the new flask.
3.2 Collagen
Preparation
and Coverslip Coating
1. Take glass-bottomed imaging dishes.
2. Place 1 mL of collagen solution at 50 μg/mL onto the glassbottomed dish for 10 min at room temperature.
3. Remove collagen solution and let dry for 1 h (see Note 3).
4. Sterilize the dishes under UV for 20 min (see Note 4).
5. Before use, wash the sterilized dishes with at least 1 mL of
DPBS, twice.
3.3 Cell Seeding
for Imaging
Seed cells 24 h before imaging. Prepare cells that express the
tension sensor as well as cells that express the tension-less control
separately. Follow steps 1–6 of the Subheading 3.1 and continue as
follows:
1. Collect the cell suspension into a sterile centrifugation tube.
2. Pellet the cells by centrifugation at 60–125 Â g for 5–8 min.
3. Meanwhile, add culture medium onto the collagen coated
imaging dishes.
4. Discard the supernatant of the centrifugation tube and
re-suspend the pellet with culture medium (see Note 5).
5. Count cells with a hematocytometer or equivalent device.
6. Plate 1:10 of the collected volume in a new flask with culture
medium.
Molecular Tension Microscopy of E-Cadherin During EMT
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Carry out all procedures in a cell culture hood unless otherwise
specified.
3.1 Cell Culture
Maintenance
Pass MDCK II cells three times a week as follows:
1. Prepare a new cell culture flask containing 3 mL of fresh culture
medium and put it into the incubator.
2. Remove old culture medium and dead cells from the cell culture flask.
3. Wash once with 1 mL of washing medium and by gently
shaking the cell culture flask.
4. Add 1 mL of Trypsin-EDTA solution in DPBS to the cell
culture flask.
5. Put the cell culture flask into incubator and wait about 10 min
for cells to detach from the surface and from each other.
6. Use a microscope to evaluate cell detachment.
7. Once cells are detached, transfer 1:10 of the collected volume
into the new flask.
3.2 Collagen
Preparation
and Coverslip Coating
1. Take glass-bottomed imaging dishes.
2. Place 1 mL of collagen solution at 50 μg/mL onto the glassbottomed dish for 10 min at room temperature.
3. Remove collagen solution and let dry for 1 h (see Note 3).
4. Sterilize the dishes under UV for 20 min (see Note 4).
5. Before use, wash the sterilized dishes with at least 1 mL of
DPBS, twice.
3.3 Cell Seeding
for Imaging
Seed cells 24 h before imaging. Prepare cells that express the
tension sensor as well as cells that express the tension-less control
separately. Follow steps 1–6 of the Subheading 3.1 and continue as
follows:
1. Collect the cell suspension into a sterile centrifugation tube.
2. Pellet the cells by centrifugation at 60–125 Â g for 5–8 min.
3. Meanwhile, add culture medium onto the collagen coated
imaging dishes.
4. Discard the supernatant of the centrifugation tube and
re-suspend the pellet with culture medium (see Note 5).
5. Count cells with a hematocytometer or equivalent device.
6. Plate 1:10 of the collected volume in a new flask with culture
medium.
Molecular Tension Microscopy of E-Cadherin During EMT
293