Cells were first fixed
with 4% paraformaldehyde (PFA) for 20 min
and then a permeabilization buffer containing phosphate-buffered saline
(PBS), 2 mg/mL Bovine Serum Albumin (BSA), and 0.1% Triton X-100.
Then, cells were incubated with phalloidin (Biotium, CA, USA) for
1 h at room temperature. Finally, cells were washed three times with
the permeabilization buffer and incubated for 2 min with 4′,6-
Diamidino-2-Phenylindol (DAPI; Sigma-Aldrich, St. Louis, MO, USA).
All coverslips were then mounted on a slide and observed under an
Olympus BX53 fluorescent microscope equipped with an XM10 Monochrome
CCD camera (Olympus Corp., Japan). For the characterization of the
actin stress fibers, the FilamentSensor tool (University of Göttingen,
Germany) was used.42 (link) In the reconstructed
images, each color corresponds to a different fiber orientation. Stress
fiber orientation was assessed using the order parameter S = cos2θ, where the higher the value of S,
the more oriented the fibers become. Cell elongation was assessed
by using optical microscopy images. ImageJ software was used to automatically
measure factor E from cells, which equals the long
axis divided by the short axis minus one. Thus, E is zero for a circle and one for an ellipse with an axis ratio of
1:2. The cells that presented E values 0–0.5
were considered as spherical, 0.5–1 as ellipsoid, and E values higher than 1 as elongated.
with 4% paraformaldehyde (PFA) for 20 min
and then a permeabilization buffer containing phosphate-buffered saline
(PBS), 2 mg/mL Bovine Serum Albumin (BSA), and 0.1% Triton X-100.
Then, cells were incubated with phalloidin (Biotium, CA, USA) for
1 h at room temperature. Finally, cells were washed three times with
the permeabilization buffer and incubated for 2 min with 4′,6-
Diamidino-2-Phenylindol (DAPI; Sigma-Aldrich, St. Louis, MO, USA).
All coverslips were then mounted on a slide and observed under an
Olympus BX53 fluorescent microscope equipped with an XM10 Monochrome
CCD camera (Olympus Corp., Japan). For the characterization of the
actin stress fibers, the FilamentSensor tool (University of Göttingen,
Germany) was used.42 (link) In the reconstructed
images, each color corresponds to a different fiber orientation. Stress
fiber orientation was assessed using the order parameter S = cos2θ, where the higher the value of S,
the more oriented the fibers become. Cell elongation was assessed
by using optical microscopy images. ImageJ software was used to automatically
measure factor E from cells, which equals the long
axis divided by the short axis minus one. Thus, E is zero for a circle and one for an ellipse with an axis ratio of
1:2. The cells that presented E values 0–0.5
were considered as spherical, 0.5–1 as ellipsoid, and E values higher than 1 as elongated.
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