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Neurite outgrowth cell-by-cell analysis using the IN Cell Developer Toolbox

th Image Analysis Module


Other materials required
Mouse renoblastoma neuro-2a (N2a) cells (ATCC)

Retinoic acid (Sigma)

Monoclonal anti-α-tubulin clone DM1A (Sigma)

Anti-mouse IgG-FITC antibody (Sigma)

Hoechst™33342 (Molecular Probes)


Assay procedure
Retinoic acid-induced neurite outgrowth was measured by quantitation of neurite extensions from fluorescently labeled neuronal cells. In the indirect immunoassay used here, FITC-conjugated secondary antibody detects anti-neurofilament 200 kD subunit (NF200) antibody. Cells were counter-stained with Hoechst 33342 to identify nuclei and imaged using the IN Cell Analyzer 3000.


Analysis procedure
1. Open image stack in the IN Cell Developer Toolbox.

2. Create target set: ‘Nuclei’
a. Select the channel with the image of the nuclei marker (Fig 2).
b. Select Preprocessing operations if required.
c. Select Segmentation option most appropriate for an accurate nuclear segmentation (Fig 3).
d. Use Erode and Sieve Postprocessing operations to refine nuclei bitmap (Fig 4).
e. Choose Count measurement and apply Statistical Function – Count to report total number of nuclei in the population.

3. Create target set: ‘Cell body’.
a. Select channel with the image of the neurite (Fig 5).
b. Select Remove Single Pixel Targets '"/>

Source:


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