3.2 Measurements
of Spiral-End
and Hook-End
Gyrations
1. Place a flow chamber on a microscope stage.
2. Observe the cells with a 100Â objective, and record the images
on a computer using a high-speed camera (see Note 4).
3. Open the recorded movie on ImageJ.
4. Duplicate the area where the cell end gyration is displayed
(Fig. 4a).
5. Set the threshold using Image > Adjust > Threshold to specify
the bending cell ends to be analyzed.
6. Set parameters in Analyze > Set Measurements > check Fit
ellipse.
7. Run analysis by Analyze > Analyze Particle, and save the result.
8. Open the result on Microsoft Excel, showing an angle versus
time plot; Fit ellipse provides the long-axis angle of the ellipse
fitted to spiral-end and hook-end (red dashed lines in Fig. 4b
and c).
9. Determine the gyration speed by analyzing peak intervals
(wavelengths) of angle-time plots or fast Fourier transform
(FFT): The wave frequency is a reciprocal of the wavelength,
and thus the gyration speed can be calculated from the average
of several peak intervals.
3.3 Observation of
PC Rotation Using
Antibody-Coated
Beads
1. Conjugate polystyrene beads with antibody by the following
procedure:
(a) Dilute 3 μL of carboxylated bead suspension (0.2 μm in
diameter) into 300 μL of 50 mM MES buffer (pH 5.2).
(b) Centrifugation at 17,000 Â g for 15 min at 23
C.
(c) Suspend the pellet in 200 μL of MES buffer and mix with
20 μL of antibody (see Note 2).
(d) Dissolve 10 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDAC) in 1 mL of MES buffer.
(e) Add 20 μL of the EDAC solution to the bead suspension
and incubate for 30 min at 23
C.
Fig. 3 A flow chamber for microscopic observation
144
Shuichi Nakamura
of Spiral-End
and Hook-End
Gyrations
1. Place a flow chamber on a microscope stage.
2. Observe the cells with a 100Â objective, and record the images
on a computer using a high-speed camera (see Note 4).
3. Open the recorded movie on ImageJ.
4. Duplicate the area where the cell end gyration is displayed
(Fig. 4a).
5. Set the threshold using Image > Adjust > Threshold to specify
the bending cell ends to be analyzed.
6. Set parameters in Analyze > Set Measurements > check Fit
ellipse.
7. Run analysis by Analyze > Analyze Particle, and save the result.
8. Open the result on Microsoft Excel, showing an angle versus
time plot; Fit ellipse provides the long-axis angle of the ellipse
fitted to spiral-end and hook-end (red dashed lines in Fig. 4b
and c).
9. Determine the gyration speed by analyzing peak intervals
(wavelengths) of angle-time plots or fast Fourier transform
(FFT): The wave frequency is a reciprocal of the wavelength,
and thus the gyration speed can be calculated from the average
of several peak intervals.
3.3 Observation of
PC Rotation Using
Antibody-Coated
Beads
1. Conjugate polystyrene beads with antibody by the following
procedure:
(a) Dilute 3 μL of carboxylated bead suspension (0.2 μm in
diameter) into 300 μL of 50 mM MES buffer (pH 5.2).
(b) Centrifugation at 17,000 Â g for 15 min at 23
C.
(c) Suspend the pellet in 200 μL of MES buffer and mix with
20 μL of antibody (see Note 2).
(d) Dissolve 10 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDAC) in 1 mL of MES buffer.
(e) Add 20 μL of the EDAC solution to the bead suspension
and incubate for 30 min at 23
C.
Fig. 3 A flow chamber for microscopic observation
144
Shuichi Nakamura