keeping the substrate clean for the healthy cells. However, this
set-up is not appropriate if experimental conditions impairs
cell–matrix adhesion as cells would detach and thus be lost.
2. Xenopus embryos can develop between 12 and 28
C, the
optimal range being 14–25
C. NC explants are a bit less
flexible and usually survive and migrate better between
18 and 21
C. Below 18
C cell–cell dispersion is affected and
explants usually remain as pseudoepithelial sheets for much
longer than they would in vivo. Above 21
C, cell survival is
lower. Between 18–21
C, explants roughly the size of a fifth of
one unilateral NC region dissected at stage 18, spread extensively during the first 2 h and then disperse as small clusters and
eventually as single cells.
3. On upright microscopes, use tight-fit lid petri dishes that can
be completely filled with culture medium and closed hermetically or open dishes with a water-immersion lens. For inverted
microscopes, any type of multi-well dishes can be used depending on the type of motorized stage. In our hands, the 8-well
Ibidi μSlides (80821) gave the best results. They allow culture
in small volumes (300 μL) suitable for drug screening. They are
made of a polymer that allows low concentration of Fibronectin coating for plastic to be used while being compatible with
confocal imaging.
4. When coating large dishes do not cover the entire dish with
Fibronectin. Use a marker pen to delimitate an area where
Fibronectin will be coated. A circle with a diameter of 1 cm is
enough to plate five rows of five explants and can be coated
with a 500 μL drop of Fibronectin 1Â solution. Always cover
the dish during Fibronectin incubation at 37
C to avoid
evaporation. If very large dishes are used (e.g., 90 mm bacteria
plates), place a piece of wet paper inside the dish to keep it
moist.
5. A successful Fibronectin coating can be assessed by eye. When
the Fibronectin solution is removed at the end of the 1-h
incubation, the part of the dish or the well that is coated
remains slightly wet and appears shiny when exposed to light
whereas a similar incubation with a drop of water or PBS1Â will
leave the dish dry as soon as the liquid is removed. The area that
received water or PBS cannot be distinguished from the rest of
the dish.
6. Dissection of Xenopus NC explants is very easy when the
embryos are at the right developmental stage. Just prior to
migration, NC cells are not significantly attached to their surrounding tissues (ectoderm, mesoderm, ectodermal placodes).
Yet, they strongly attach to each other. This allows the whole
NC region to be removed, as it is an epithelial structure only
268
Nade ` ge Gouignard et al.
set-up is not appropriate if experimental conditions impairs
cell–matrix adhesion as cells would detach and thus be lost.
2. Xenopus embryos can develop between 12 and 28
C, the
optimal range being 14–25
C. NC explants are a bit less
flexible and usually survive and migrate better between
18 and 21
C. Below 18
C cell–cell dispersion is affected and
explants usually remain as pseudoepithelial sheets for much
longer than they would in vivo. Above 21
C, cell survival is
lower. Between 18–21
C, explants roughly the size of a fifth of
one unilateral NC region dissected at stage 18, spread extensively during the first 2 h and then disperse as small clusters and
eventually as single cells.
3. On upright microscopes, use tight-fit lid petri dishes that can
be completely filled with culture medium and closed hermetically or open dishes with a water-immersion lens. For inverted
microscopes, any type of multi-well dishes can be used depending on the type of motorized stage. In our hands, the 8-well
Ibidi μSlides (80821) gave the best results. They allow culture
in small volumes (300 μL) suitable for drug screening. They are
made of a polymer that allows low concentration of Fibronectin coating for plastic to be used while being compatible with
confocal imaging.
4. When coating large dishes do not cover the entire dish with
Fibronectin. Use a marker pen to delimitate an area where
Fibronectin will be coated. A circle with a diameter of 1 cm is
enough to plate five rows of five explants and can be coated
with a 500 μL drop of Fibronectin 1Â solution. Always cover
the dish during Fibronectin incubation at 37
C to avoid
evaporation. If very large dishes are used (e.g., 90 mm bacteria
plates), place a piece of wet paper inside the dish to keep it
moist.
5. A successful Fibronectin coating can be assessed by eye. When
the Fibronectin solution is removed at the end of the 1-h
incubation, the part of the dish or the well that is coated
remains slightly wet and appears shiny when exposed to light
whereas a similar incubation with a drop of water or PBS1Â will
leave the dish dry as soon as the liquid is removed. The area that
received water or PBS cannot be distinguished from the rest of
the dish.
6. Dissection of Xenopus NC explants is very easy when the
embryos are at the right developmental stage. Just prior to
migration, NC cells are not significantly attached to their surrounding tissues (ectoderm, mesoderm, ectodermal placodes).
Yet, they strongly attach to each other. This allows the whole
NC region to be removed, as it is an epithelial structure only
268
Nade ` ge Gouignard et al.