When performing invasion and migration assays, the Scratch Wound scan type can be
utilized at any of the three objectives (4X, 10X, or 20X). Additionally, a “Wide Mode” scan
type is available to ensure that both boundaries of the wound are imaged. The “Wide
Mode” scan type is mandatory when acquiring images using the 20X objective, is optional
when using the 10X objective, and is not an available feature when scanning vessels at
4X. To utilize the “Wide Mode” scan type, select the check box located beneath the scan
type in the “Scan Mode” task pane.
8.2 Cell Migration/Invasion Module
The Cell Migration/Invasion Software Module is used to identify the Relative Wound Density of
cells invading or migrating into a scratch created using the Essen Woundmaker™. Depending on
your type of assay, the IncuCyte™ ZOOM used in conjunction with the Cell Migration/Invasion
Module can measure the rate and extent of cell movement into that wound over time. In the first
step of the process, the cell migration/invasion algorithm analyzes each image and assigns a well-
specific wound mask that corresponds to the Initial Scratch Wound. From here a Scratch Wound
Mask follows invading and migrating cells as they move into the initial wound, creating a
measurement of Relative Wound Density.
8.3 Angiogenesis Analysis Module
The Angiogenesis Analysis Software Module is used to identify the fluorescent signal from
background in order to quantify multiple angiogenic assay metrics, such as tube length and
branch formation, for each time point. In the first step of the process, the angiogenesis algorithm
analyzes each fluorescent image and assigns a segmentation mask that closely resembles the in
vitro network. From here, the mask can be refined and filtered to exclude non-tube forming
events, specifically measuring angiogenesis over time.
8.3.1 Tiled Field of View Mosaic Imaging Mode
The Tiled Field of View (FOV) mosaic image scan type stitches together six images, from a
2 x 3 grid, to produce a larger, more integrated view of the angiogenic network (Figure
30). Because the scanning process acquires 6 images per well, the scan length will be
longer than that for a plate run using the 4X objective.
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