A simple method for precisely controlling the confinement of cells in culture
Within the confines of tissues, cells rarely migrate by a single mechanism. Recent reports have revealed a transition from a mesenchymal to a bleb-based form of motility occurs when cells are confined. Until now, control over the amount of confinement in in vitro tissue culture required the use of microfabrication techniques. This protocol describes seven easy steps to assemble a simple system that allows control of the degree of cell confinement and the adhesivity of the cellular microenvironment in culture conditions that allow analysis of cell behavior by high-resolution light microscopic imaging. This method requires only basic lab tissue culture equipment and thus should make studies on the effects of confinement on cellular processes including cell migration, division and differentiation, accessible to a broad range of researchers. We validate that the method can precisely control confinement and that high confinement of human melanoma A375 cells promotes leader bleb-based migration.
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Movie S2 High confinement will promote positioning of the nucleus inside the leader bleb and leader bleb-based migration Central Z-plane confocal time-lapse movie of leader bleb-based migration in an A375 cell that was co-expressing Emerald-F-tractin (red, actin cytoskeleton) and FusionRed-H2B (green, nucleus), confined between PDMS and BSA coated glass maintained at h= 3.11 µm by polystyrene beads. Note that the speed of migration increases after the nucleus enters the leader bleb, like the “A2” phenotype previously described by Liu et al. (2015). Scale bar and elapsed time are shown.
Movie S1 Low confinement of cells will not promote leader bleb-based migration Central Z-plane confocal time-lapse movie of continuous blebbing in an A375 cell that was co-expressing Emerald-F-tractin (red, actin cytoskeleton) and FusionRed-H2B (green, nucleus), confined between PDMS and BSA coated glass maintained at h=5.15 µm by polystyrene beads. Scale bar and elapsed time are shown.
Posted 12 Feb, 2018
A simple method for precisely controlling the confinement of cells in culture
Posted 12 Feb, 2018
Within the confines of tissues, cells rarely migrate by a single mechanism. Recent reports have revealed a transition from a mesenchymal to a bleb-based form of motility occurs when cells are confined. Until now, control over the amount of confinement in in vitro tissue culture required the use of microfabrication techniques. This protocol describes seven easy steps to assemble a simple system that allows control of the degree of cell confinement and the adhesivity of the cellular microenvironment in culture conditions that allow analysis of cell behavior by high-resolution light microscopic imaging. This method requires only basic lab tissue culture equipment and thus should make studies on the effects of confinement on cellular processes including cell migration, division and differentiation, accessible to a broad range of researchers. We validate that the method can precisely control confinement and that high confinement of human melanoma A375 cells promotes leader bleb-based migration.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
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