10. When plating explants next to each other onto Fibronectin, do
not put explants to close to one another as NC cells can attract
each other via a process called co-attraction or mutual attraction. This would lead neighboring explants to merge. A rule of
thumb is to make sure that each explant is at least at five times
its own diameter away from the nearest explants in the dish.
11. If only overall explant dispersion is to be analyzed, time-lapse
imaging is not necessary. Explants can be fixed after several
hours and the dispersion can be analyzed using triangulation,
total explant area or nearest neighbors analysis. However, having the dynamics of the population and the movement of single
cells can be very informative. For movies done with a 10Â
objective at a temperature between 18–21
C, cells migrate at
around 1–3 μm/min and a time interval of 3 min is optimal for
cell tracking (either manual or automated). Eight-hour movies
at 21
C are enough to see the main steps of dispersion:
adhesion, spreading, break-up of the explants as clusters and
single cell dispersion. If under control conditions single cell
dispersion does not occur within this time frame it can be due
to several possibilities: (i) the temperature is too low, (ii) cells
were extracted at earlier stages than 18 and have not yet completed their cadherin switch (there is a sharp decrease of the
amount of E-cadherin after stage 15), (iii) the substrate is not
optimal for migration and cells have problems moving away
from each other.
12. To fix cells at the end of a culture experiments do not remove
the DFA1Â, instead add directly formaldehyde into the dish.
In 8-well slides, if each well contains 300 μL of DFA1X, simply
add 100 μL of formaldehyde 16% in each well. In larger dishes,
add locally formaldehyde 16% atop the explants to pre-fix the
cells, wait 5 min, remove the medium, fill with formaldehyde
4%, and further fix for 30 min.
13. To label NC cells prior to cell culture, one can inject at either
2, 4, 8, 16, or 32-cell stage. If simple tracers to label nuclei
and/or membranes are used, the easiest way is to inject two
blastomeres at 2-cell stage and select the brightest embryos at
stage 16 prior to removing the vitelline membrane. If nuclear/
membrane tracers are to be used in combination with other
treatments that may affect early development, it is recommended to inject at 8-cell stage or later. From 8-cell stage the
animal blastomeres only contribute to the ectoderm (neural
plate, NC, placodes, epidermis), therefore injections at 8-cell
stage or later will leave the mesoderm and endoderm intact,
reducing possible side effects on gastrulation.
14. In control conditions, cell–cell dissociation leads to a progressive shift from collective/pseudoepithelial-like behavior, to a
collective/mesenchymal behavior, to single cell behavior.
Using Xenopus Neural Crest to Study EMT
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not put explants to close to one another as NC cells can attract
each other via a process called co-attraction or mutual attraction. This would lead neighboring explants to merge. A rule of
thumb is to make sure that each explant is at least at five times
its own diameter away from the nearest explants in the dish.
11. If only overall explant dispersion is to be analyzed, time-lapse
imaging is not necessary. Explants can be fixed after several
hours and the dispersion can be analyzed using triangulation,
total explant area or nearest neighbors analysis. However, having the dynamics of the population and the movement of single
cells can be very informative. For movies done with a 10Â
objective at a temperature between 18–21
C, cells migrate at
around 1–3 μm/min and a time interval of 3 min is optimal for
cell tracking (either manual or automated). Eight-hour movies
at 21
C are enough to see the main steps of dispersion:
adhesion, spreading, break-up of the explants as clusters and
single cell dispersion. If under control conditions single cell
dispersion does not occur within this time frame it can be due
to several possibilities: (i) the temperature is too low, (ii) cells
were extracted at earlier stages than 18 and have not yet completed their cadherin switch (there is a sharp decrease of the
amount of E-cadherin after stage 15), (iii) the substrate is not
optimal for migration and cells have problems moving away
from each other.
12. To fix cells at the end of a culture experiments do not remove
the DFA1Â, instead add directly formaldehyde into the dish.
In 8-well slides, if each well contains 300 μL of DFA1X, simply
add 100 μL of formaldehyde 16% in each well. In larger dishes,
add locally formaldehyde 16% atop the explants to pre-fix the
cells, wait 5 min, remove the medium, fill with formaldehyde
4%, and further fix for 30 min.
13. To label NC cells prior to cell culture, one can inject at either
2, 4, 8, 16, or 32-cell stage. If simple tracers to label nuclei
and/or membranes are used, the easiest way is to inject two
blastomeres at 2-cell stage and select the brightest embryos at
stage 16 prior to removing the vitelline membrane. If nuclear/
membrane tracers are to be used in combination with other
treatments that may affect early development, it is recommended to inject at 8-cell stage or later. From 8-cell stage the
animal blastomeres only contribute to the ectoderm (neural
plate, NC, placodes, epidermis), therefore injections at 8-cell
stage or later will leave the mesoderm and endoderm intact,
reducing possible side effects on gastrulation.
14. In control conditions, cell–cell dissociation leads to a progressive shift from collective/pseudoepithelial-like behavior, to a
collective/mesenchymal behavior, to single cell behavior.
Using Xenopus Neural Crest to Study EMT
271