6. Go to “analyze/tools/ROI manager” and do the following
steps: “Add”, “Split”, “multi-measures.”
7. Save the results table containing the areas of all triangles. It is
important to note that the first value of that list is the cumulated area occupied by all triangles (see Note 15).
8. If this has to be done on multiple images, a macro can be used.
A macro for batch processing and a custom-made Look-up
table (LUT) for color-coding of triangles were developed in
the Theveneau lab and are available upon request.
The respective merits of the two methods to analyze dispersion
are discussed in Note 16.
4 Notes
1. In Xenopus NC cells the dynamics of dispersion depends on
the timing of cell–cell dissociation, which is linked to the
E-to-N-cadherin switch. Thus, the timing (how long it takes)
and intensity (are all cells becoming individual?) of dispersion
are a proxy for EMT dynamics. This can be assessed on low
magnification images even without time-lapse if several conditions are plated at the same t0 and compared at a similar time
point after the beginning of the cell culture. Therefore, monitoring simultaneously several explants at low magnification on
a dissecting microscope could be sufficient for a first screening
of experimental conditions. However, if dispersion analysis is to
be performed using single cell detection by nuclear staining or
if cell tracking is to be done in parallel, then higher magnifications (in XY and time) are required and a motorized stage is
necessary. A wide field microscope with a 10Â objective will
allow visualization of NC explants the size of a fifth of a
complete unilateral NC region, while providing enough XY
resolution to track single cells accurately using a time interval of
3 min. If cells are labelled with a membrane tracer and the
dynamics of cell–cell contacts is to be analyzed, 20Â or 40Â
objectives are needed and shorter time intervals are necessary
(1 min or below). Inverted microscopes are the usual choice for
cell culture assays. However, we obtained very good results
using upright microscopes with dry lenses. In this case, cells
need to be cultured in a dish that can be filled entirely with
culture medium, without any air bubbles, and flipped upside
down. Several providers have culture dishes with tight lids in
their catalogue. If your cell culture device of choice does not
exist in a version with a tight-fit lid, it is possible to seal the lid
using high vacuum silicone grease. This type of upside-down
cell culture removes all dead and/or non-adherent cells,
Using Xenopus Neural Crest to Study EMT
267
steps: “Add”, “Split”, “multi-measures.”
7. Save the results table containing the areas of all triangles. It is
important to note that the first value of that list is the cumulated area occupied by all triangles (see Note 15).
8. If this has to be done on multiple images, a macro can be used.
A macro for batch processing and a custom-made Look-up
table (LUT) for color-coding of triangles were developed in
the Theveneau lab and are available upon request.
The respective merits of the two methods to analyze dispersion
are discussed in Note 16.
4 Notes
1. In Xenopus NC cells the dynamics of dispersion depends on
the timing of cell–cell dissociation, which is linked to the
E-to-N-cadherin switch. Thus, the timing (how long it takes)
and intensity (are all cells becoming individual?) of dispersion
are a proxy for EMT dynamics. This can be assessed on low
magnification images even without time-lapse if several conditions are plated at the same t0 and compared at a similar time
point after the beginning of the cell culture. Therefore, monitoring simultaneously several explants at low magnification on
a dissecting microscope could be sufficient for a first screening
of experimental conditions. However, if dispersion analysis is to
be performed using single cell detection by nuclear staining or
if cell tracking is to be done in parallel, then higher magnifications (in XY and time) are required and a motorized stage is
necessary. A wide field microscope with a 10Â objective will
allow visualization of NC explants the size of a fifth of a
complete unilateral NC region, while providing enough XY
resolution to track single cells accurately using a time interval of
3 min. If cells are labelled with a membrane tracer and the
dynamics of cell–cell contacts is to be analyzed, 20Â or 40Â
objectives are needed and shorter time intervals are necessary
(1 min or below). Inverted microscopes are the usual choice for
cell culture assays. However, we obtained very good results
using upright microscopes with dry lenses. In this case, cells
need to be cultured in a dish that can be filled entirely with
culture medium, without any air bubbles, and flipped upside
down. Several providers have culture dishes with tight lids in
their catalogue. If your cell culture device of choice does not
exist in a version with a tight-fit lid, it is possible to seal the lid
using high vacuum silicone grease. This type of upside-down
cell culture removes all dead and/or non-adherent cells,
Using Xenopus Neural Crest to Study EMT
267