Remove the PBS. Immerse the cells in 1 mL trypsin and store at
37
C for 5 min.
8. Check the cells are in suspension using an optical microscope.
Transport the cells into a 5 mL Eppendorf tube. Centrifuge the
cells at 180 Â g for 5 min to obtain a cell pellet.
9. Remove the excess medium. Add 100 μL cell culture medium
and resuspend the cells.
10. Seed the cells onto the gelatin fibers patterned on PDMS to
obtain a coverage of 100 cells/mm
2 (see Note 9). Incubate the
cells at 37
C, 5% CO 2 .
3.3 Live-Cell
Imaging
1. Turn on the live cell imaging chamber. Set the temperature to
37
C and turn on the CO 2 supply. Wait 30 min for the
temperature and CO 2 level to stabilize.
2. Turn on the confocal microscope and 633 nm laser, acquire in
the transmission mode.
3. Place the sample petri-dish into the chamber. Top up the
medium if necessary.
4. Adjust the focus of the confocal microscope. Adjust the laser
power and digital gain to obtain sharp images. The laser power
should be as low as possible to avoid damage to the cells. Find
the region of interest.
5. Set the scanning rate to 400 Hz, and image size to 512 Â 512
pixels. Set the acquisition time to 12 h with 10 min intervals.
6. Start imaging. A sequence of live cell images, indicating the
interactions between EA.hy926 cells with gelatin fibers patterned on PDMS, is shown in Fig. 4a–e.
3.4 Immunofluorescent Staining
1. Immerse the sample in the fixation solution (4% formaldehyde
in PBS) for 20 min.
2. Wash the sample twice with the buffer solution (0.05% Tween20 in PBS).
3. Permeabilize the cells with 0.1% Triton X-100 solution in PBS
for 5 min.
4. Wash the sample twice with the buffer solution (0.05% Tween20 in PBS).
5. Immerse the sample in the blocking solution (1% BSA in PBS)
for 30 min.
6. Remove the blocking solution.
7. Add phalloidin (1:400 dilution) and Hoechst (1:10000 dilution) in PBS. Incubate the sample in this solution for 50 min.
8. Wash the sample three times with the buffer solution. Leave the
sample in the solution for 5 min during each wash.
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37
C for 5 min.
8. Check the cells are in suspension using an optical microscope.
Transport the cells into a 5 mL Eppendorf tube. Centrifuge the
cells at 180 Â g for 5 min to obtain a cell pellet.
9. Remove the excess medium. Add 100 μL cell culture medium
and resuspend the cells.
10. Seed the cells onto the gelatin fibers patterned on PDMS to
obtain a coverage of 100 cells/mm
2 (see Note 9). Incubate the
cells at 37
C, 5% CO 2 .
3.3 Live-Cell
Imaging
1. Turn on the live cell imaging chamber. Set the temperature to
37
C and turn on the CO 2 supply. Wait 30 min for the
temperature and CO 2 level to stabilize.
2. Turn on the confocal microscope and 633 nm laser, acquire in
the transmission mode.
3. Place the sample petri-dish into the chamber. Top up the
medium if necessary.
4. Adjust the focus of the confocal microscope. Adjust the laser
power and digital gain to obtain sharp images. The laser power
should be as low as possible to avoid damage to the cells. Find
the region of interest.
5. Set the scanning rate to 400 Hz, and image size to 512 Â 512
pixels. Set the acquisition time to 12 h with 10 min intervals.
6. Start imaging. A sequence of live cell images, indicating the
interactions between EA.hy926 cells with gelatin fibers patterned on PDMS, is shown in Fig. 4a–e.
3.4 Immunofluorescent Staining
1. Immerse the sample in the fixation solution (4% formaldehyde
in PBS) for 20 min.
2. Wash the sample twice with the buffer solution (0.05% Tween20 in PBS).
3. Permeabilize the cells with 0.1% Triton X-100 solution in PBS
for 5 min.
4. Wash the sample twice with the buffer solution (0.05% Tween20 in PBS).
5. Immerse the sample in the blocking solution (1% BSA in PBS)
for 30 min.
6. Remove the blocking solution.
7. Add phalloidin (1:400 dilution) and Hoechst (1:10000 dilution) in PBS. Incubate the sample in this solution for 50 min.
8. Wash the sample three times with the buffer solution. Leave the
sample in the solution for 5 min during each wash.
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Zhaoying Li et al.