15. Live imaging of aggregates can begin at 2 hpa after all cells in
the PCR tube are adherent and form an aggregate.
16. The aggregate can then be mounted in a glass-bottomed culture dish [43]. To reduce drift during imaging, aggregates can
positioned either in an EM grid or skewered with a filament cut
from a length of heat-pulled plastic pipette tip. The diameter of
the filament skewer can range between 50 and 100 μm (see
Note 8).
17. Set the confocal acquisition to collect a Z-stack to acquire the
images of spherical shaped aggregates over-time. Z-intervals,
e.g., slice spacing, should be set to allow appropriate 3D
reconstruction and image analysis. Live cell imaging allows
Fig. 2 Imaging mesenchymal-to-epithelial transition in deep ectoderm aggregates (a) Schematic representation of steps to make aggregates. Ectoderm explants with superficial epithelial (orange) and deep mesenchymal cell layers (blue) are isolated and separated. After 2 h of aggregation at the bottom of the non-adherent
PCR tube, the spheroidal aggregates can be subject to live imaging of the epithelialization. (b) (I–III)
Representative frames from time-lapse sequences showing expanding epithelial patches from 2 to 6 hpa.
Patches expand by recruiting GFP-ZO-1 positive cells to the edge of existing patch. Row I depicts images, row
II depicts lookup-table-inverted images where MET cells have been outlined in red, and in row III different
colors indicate cell tracking within a growing patch. Scale bar, 50 μm. (c) Surface area of the epithelial cell
patch (from b) continues to increase, although the number of cells within that patch is unchanged after 5 hpa.
Note: Patches can continue to add cells beyond this time. (d) Surface area of individual cells (from b I–III)
increase more than twofold over 1–2 h. (e) Top row shows representative frames from time-sequences
showing MET spreads by joining patches of GFP-ZO-1 positive cells. Lower row indicates MET cells outlined in
red on LUT-inverted images. Scale bar, 50 μm
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Hye Young Kim and Lance A. Davidson
the PCR tube are adherent and form an aggregate.
16. The aggregate can then be mounted in a glass-bottomed culture dish [43]. To reduce drift during imaging, aggregates can
positioned either in an EM grid or skewered with a filament cut
from a length of heat-pulled plastic pipette tip. The diameter of
the filament skewer can range between 50 and 100 μm (see
Note 8).
17. Set the confocal acquisition to collect a Z-stack to acquire the
images of spherical shaped aggregates over-time. Z-intervals,
e.g., slice spacing, should be set to allow appropriate 3D
reconstruction and image analysis. Live cell imaging allows
Fig. 2 Imaging mesenchymal-to-epithelial transition in deep ectoderm aggregates (a) Schematic representation of steps to make aggregates. Ectoderm explants with superficial epithelial (orange) and deep mesenchymal cell layers (blue) are isolated and separated. After 2 h of aggregation at the bottom of the non-adherent
PCR tube, the spheroidal aggregates can be subject to live imaging of the epithelialization. (b) (I–III)
Representative frames from time-lapse sequences showing expanding epithelial patches from 2 to 6 hpa.
Patches expand by recruiting GFP-ZO-1 positive cells to the edge of existing patch. Row I depicts images, row
II depicts lookup-table-inverted images where MET cells have been outlined in red, and in row III different
colors indicate cell tracking within a growing patch. Scale bar, 50 μm. (c) Surface area of the epithelial cell
patch (from b) continues to increase, although the number of cells within that patch is unchanged after 5 hpa.
Note: Patches can continue to add cells beyond this time. (d) Surface area of individual cells (from b I–III)
increase more than twofold over 1–2 h. (e) Top row shows representative frames from time-sequences
showing MET spreads by joining patches of GFP-ZO-1 positive cells. Lower row indicates MET cells outlined in
red on LUT-inverted images. Scale bar, 50 μm
282
Hye Young Kim and Lance A. Davidson