Page 105 - Live-cellanalysis handbook

Kinetic Neurite Analysis Assays

Sample results

Automated analysis and visualization in label-free monocultures
The IncuCyte Live-Cell Analysis System takes non-invasive phase- processes such as neurite initiation, neurite extension, branching,
contrast images of neuronal cultures for a complete time course and loss of neurite length due to retraction or disintegration.
of neurite dynamics (Figure 1, top image). NeuroTrack software Quantification of cell body cluster count and area provides
produces data on neurite dynamics by analyzing these images. methods to measure cell body size or proliferation, as well as
The software analyzes a HD phase image of neurons in two steps: providing two metrics for normalization.
1) Cell bodies are segmented from background based on texture
and/or brightness, and are masked as cell body clusters (Figure 1, To validate the data generated provided by the NeuroTrack
lower left). The number of clusters and the total area of clusters software, data from a live-cell IncuCyte Neurite Analysis assay was
are normalized to image area. 2) Linear features are detected compared to end-point data derived from fixed cells imaged on
based on width and brightness, and are masked as neurites (Figure a high content screening system (Figure 2). At the three different
1, lower right). The total neurite length and the total number of time points, NeuroTrack’s automated software quantified neurite
branch points are normalized to image area. The segmentation length in unfixed cells over the course of a 6.5 day assay with
mask can be refined and filtered to tailor the mask to the specific a sensitivity statistically identical to the fixing/immunostaining
cell type. Every time point of the assay generates metrics from the method quantified using a high content screening system.
automated software analysis, which quantifies biologically relevant

Neurite length
per mm 2
40

IXM-immuno-
30 fluorescence
Live-cell
imaging
20

10

0
18 90 162
Time post plating (h)
Figure 2: Comparison of automated IncuCyte NeuroTrack analysis to
traditional fixed-point staining. Three 96- well plates from the same E18
rat cortical prep were seeded at 8,000 cells/well. One plate was imaged in
the IncuCyte at each of three different time points post-plating (18, 90,
and 162 hours). These images were quantified with IncuCyte NeuroTrack to
measure neurite length/mm . Immediately after imaging in an IncuCyte ,
2
®
plates were removed from the incubator, fixed, and immunostained for
β-tubulin to mark the neurite structures. Cells were then imaged in
Figure 1. Analysis of rat E18 cortical neurons structures using label-free an Image Xpress Micro high-content screening system and MetaXpress
segmentation masking. Phase image of neurons (top) after 5 days in vitro software was used to quantify neurite length/mm . The two data sets
2
(DIV). Cell body cluster mask applied to image (bottom left). Neurite mask are statically indistinguishable at each time point. Error bars represent
and cell body cluster mask applied to image (bottom right). standard deviation, N=6.

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