3.3 Analysis of Cell
Migration
and Dispersion
Dynamics
3.3.1 Analysis of Cell
Migration
1. A nuclear tracer (e.g., H2B-mCherry/GFP) must be injected
into early Xenopus embryos prior to dissection if single cell
tracking is to be performed (see Note 13). Manual or automated tracking can then be done using ImageJ/FIJI (Manual
tracking or trackmate plug-in) or commercial alternatives such
as Imaris/Bitplane (spot tracking algorithm).
2. From tracks, cell speed and directionality can be retrieved. This
information can then be correlated with the dynamics of cell
dispersion (see Note 14).
3.3.2 Analysis of Cell
Dispersion Using the Total
Explored Area Over Time
(Fig. 4)
1. In ImageJ/FIJI use the “image\stacks\tools\make substack”
tool to extract images from each movie at regular interval
(e.g., first image and then one image per hour). Save all substacks (one per XY position) into a separate folder for analysis
(Fig. 4a).
2. In ImageJ/FIJI, link all external cells of an explant using the
broken line tool and measure the area. Repeat this step for each
time step of the substack (Fig. 4b, c). Repeat for each substack
movie. Save the results as .xls or tabulated text file.
3. If only one experimental condition is analyzed over time, plotting the data as box whiskers plots for each time point will allow
variability at each time point and overall dispersion over time to
be assessed. If multiple conditions are to be plotted in parallel
on the same graph, curves representing the mean area per time
point alongside the standard deviation are more practical.
3.3.3 Analysis of Cell
Dispersion Using
Triangulation Between
Nearest Neighbors
1. In ImageJ/FIJI use the “image\stacks\tools\make substack”
tool to extract images from each movie at regular interval
(e.g., first image and then one image per hour). Save all substacks (one per XY position) into a separate folder for analysis.
2. In imageJ/FIJI, go to “analyze/set measurements” and make
sure “centroid” and “area” are selected.
3. Open one of the substacks and estimate the mean area occupied
by one nucleus in your samples by measuring about 20 different
nuclei using the elliptical or polygon selection tools in ImageJ/
FIJI.
4. Go to “analyze/analyze particles.” Set the minimum and maximum area occupied by a nucleus as measured in step 3 and
press OK. This will produce a list of XY coordinates of all nuclei
detected.
5. Use the Delaunay/Voronoi plug-in in ImageJ/FIJI to build
triangles from coordinates produced by the “analyze particles”
tool. The options “infer selection from particles” and “make
Delaunay ROI” must be selected.
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Migration
and Dispersion
Dynamics
3.3.1 Analysis of Cell
Migration
1. A nuclear tracer (e.g., H2B-mCherry/GFP) must be injected
into early Xenopus embryos prior to dissection if single cell
tracking is to be performed (see Note 13). Manual or automated tracking can then be done using ImageJ/FIJI (Manual
tracking or trackmate plug-in) or commercial alternatives such
as Imaris/Bitplane (spot tracking algorithm).
2. From tracks, cell speed and directionality can be retrieved. This
information can then be correlated with the dynamics of cell
dispersion (see Note 14).
3.3.2 Analysis of Cell
Dispersion Using the Total
Explored Area Over Time
(Fig. 4)
1. In ImageJ/FIJI use the “image\stacks\tools\make substack”
tool to extract images from each movie at regular interval
(e.g., first image and then one image per hour). Save all substacks (one per XY position) into a separate folder for analysis
(Fig. 4a).
2. In ImageJ/FIJI, link all external cells of an explant using the
broken line tool and measure the area. Repeat this step for each
time step of the substack (Fig. 4b, c). Repeat for each substack
movie. Save the results as .xls or tabulated text file.
3. If only one experimental condition is analyzed over time, plotting the data as box whiskers plots for each time point will allow
variability at each time point and overall dispersion over time to
be assessed. If multiple conditions are to be plotted in parallel
on the same graph, curves representing the mean area per time
point alongside the standard deviation are more practical.
3.3.3 Analysis of Cell
Dispersion Using
Triangulation Between
Nearest Neighbors
1. In ImageJ/FIJI use the “image\stacks\tools\make substack”
tool to extract images from each movie at regular interval
(e.g., first image and then one image per hour). Save all substacks (one per XY position) into a separate folder for analysis.
2. In imageJ/FIJI, go to “analyze/set measurements” and make
sure “centroid” and “area” are selected.
3. Open one of the substacks and estimate the mean area occupied
by one nucleus in your samples by measuring about 20 different
nuclei using the elliptical or polygon selection tools in ImageJ/
FIJI.
4. Go to “analyze/analyze particles.” Set the minimum and maximum area occupied by a nucleus as measured in step 3 and
press OK. This will produce a list of XY coordinates of all nuclei
detected.
5. Use the Delaunay/Voronoi plug-in in ImageJ/FIJI to build
triangles from coordinates produced by the “analyze particles”
tool. The options “infer selection from particles” and “make
Delaunay ROI” must be selected.
266
Nade ` ge Gouignard et al.