tracking of MET in single cells and the emergences of large
patches of epithelial clusters (Fig. 2b). Following individual
cells over time allow quantitation of cell number and areas as
the patches emerge and grow in size (Fig. 2c–d). Live cell
ismallermaging also reveals growth of epithelial sheets by
fusion of multiple small clusters (Fig. 2e).
4 Notes
1. For live imaging of aggregates, a custom fabricated acrylic well
can be glued on to a large cover glass to build a glass-bottomed
imaging chamber using silicon grease. Custom sized chamber
can be milled by a standard machine shop or fabricated by a 3D
FDM printer. The volume of custom imaging chambers can be
reduced to limit amounts of small molecule inhibitors or limited availability reagents such as function blocking antibodies.
Chambers fashioned from milled acrylic can be reused after
thorough washing with 70% ethanol and water. Commercial
multi-well chambers are suited for multiple non-imaging
experimental conditions. For Imaging of fixed aggregates,
inexpensive disposable imaging chambers can be constructed
with nylon washer (inner diameter 8.5–13 mm) glued to cover
glass with silicon grease (for aqueous mount only) or fingernail
polish (required to be resistant to Murray’s clear). For the most
stable imaging, the top of the chamber should be sealed with an
appropriately sized circular cover glass. Variable size and thickness of washers are available through common tool and hardware suppliers (e.g., Grainger Industrial Supply).
2. Cells in the epithelial superficial layer are more tightly bound
and are more resistant to dissociation than deep cell layers. This
intrinsic property and color of darkly pigmented cells in the
superficial layer can be used to distinguish superficial cells from
less-pigmented deep cells during preparation of routine aggregates, however, before relying on this routine practice we recommend confirming superficial cells are not contaminating
aggregates. To confirm the effectiveness of microsurgery and
dissociation we recommend carrying out aggregate assembly
using embryos whose surface layers have been labeled with a
fluorescent tag. To label the surface layer we take stage
9 embryos and label free prolines on apical proteins with
NHS-rhodamine. Incubate embryos in NHS-rhodamine
(~1 μg/ml) in elevated pH (>9) 1/3Â MBS for 30 min on a
slow operating rotator. Wash embryos three times with 1/3Â
MBS. This produces embryos with a covalent fluorescent label
on the apical cell surface can be easily detected within deep
ectoderm aggregates if contaminated with cells from superficial
layer.
Xenopus Deep Cell Aggregates: A 3D Model for MET
283
patches of epithelial clusters (Fig. 2b). Following individual
cells over time allow quantitation of cell number and areas as
the patches emerge and grow in size (Fig. 2c–d). Live cell
ismallermaging also reveals growth of epithelial sheets by
fusion of multiple small clusters (Fig. 2e).
4 Notes
1. For live imaging of aggregates, a custom fabricated acrylic well
can be glued on to a large cover glass to build a glass-bottomed
imaging chamber using silicon grease. Custom sized chamber
can be milled by a standard machine shop or fabricated by a 3D
FDM printer. The volume of custom imaging chambers can be
reduced to limit amounts of small molecule inhibitors or limited availability reagents such as function blocking antibodies.
Chambers fashioned from milled acrylic can be reused after
thorough washing with 70% ethanol and water. Commercial
multi-well chambers are suited for multiple non-imaging
experimental conditions. For Imaging of fixed aggregates,
inexpensive disposable imaging chambers can be constructed
with nylon washer (inner diameter 8.5–13 mm) glued to cover
glass with silicon grease (for aqueous mount only) or fingernail
polish (required to be resistant to Murray’s clear). For the most
stable imaging, the top of the chamber should be sealed with an
appropriately sized circular cover glass. Variable size and thickness of washers are available through common tool and hardware suppliers (e.g., Grainger Industrial Supply).
2. Cells in the epithelial superficial layer are more tightly bound
and are more resistant to dissociation than deep cell layers. This
intrinsic property and color of darkly pigmented cells in the
superficial layer can be used to distinguish superficial cells from
less-pigmented deep cells during preparation of routine aggregates, however, before relying on this routine practice we recommend confirming superficial cells are not contaminating
aggregates. To confirm the effectiveness of microsurgery and
dissociation we recommend carrying out aggregate assembly
using embryos whose surface layers have been labeled with a
fluorescent tag. To label the surface layer we take stage
9 embryos and label free prolines on apical proteins with
NHS-rhodamine. Incubate embryos in NHS-rhodamine
(~1 μg/ml) in elevated pH (>9) 1/3Â MBS for 30 min on a
slow operating rotator. Wash embryos three times with 1/3Â
MBS. This produces embryos with a covalent fluorescent label
on the apical cell surface can be easily detected within deep
ectoderm aggregates if contaminated with cells from superficial
layer.
Xenopus Deep Cell Aggregates: A 3D Model for MET
283