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

B>option in the Target Details panel.
c. Select Preprocessing operations if required.
d. Select Segmentation option that will identify whole cells (cell bodies and neurites) (Fig 6).
e. Use Erode operation to exclude neurites from the bitmap (Fig 7).
f. Use the Sieve operation to filter out debris mainly remaining from the joint points.
g. Use the Dilate operation with the same kernel settings as the previous Erode step to restore the cell bodies bitmap so that the outer edge of the mask coincides with the cell bodies edge but no neurites are included (Fig 8).
h. Use a second Dilate step with minimal kernel size in order to overlap the cell bodies bitmap with the neurites bitmap.
i. Apply Clump Breaking operation using ‘Nuclei’ as ‘seed’ (Fig 9).
j. Choose Count measurement and apply Statistical Function - Count to report total number of cell bodies in the population.

4. Create target set: ‘Neurites’.
a. Select channel with the image of neurites (Fig 5).
b. Create a macro that includes the following operation: Subtraction of the dilated cell bodies bitmap (Step 3g) from the whole cells bitmap (Step 3d). Include this macro in the protocol as ‘Preprocessing Macro’.
c. Select Intensity Segmentation option to create a mask from the output of the preprocessing operation by setting the lower intensity threshold above 0 (Fig 10).

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