Table 1.

Description of each module of the CellProfiler pipeline

Module	Description
IdentifyPrimaryObjects	Identifies the nuclei via Hoechst staining. Determine the typical diameter empirically by testing different image sets. Nuclei touching the border of the image are removed.
Threshold	Converts the SMI312 grayscale image into a binary image for masking the nuclei in the next step.
MaskObjects	Keeps only those nuclei with at least 20% overlap with the SMI312 staining. This way only neuronal nuclei will remain. This might be necessary for primary cultures with a mixed assortment of cell types.
IdentifySecondaryObjects	Identifies axons (+cell body) in the SMI312 image around the previously identified and masked nuclei.
IdentifySecondaryObjects	Identify dendrites (+cell body) by using the MAP2 image around the previously identified and masked nuclei.
MaskObjects	Both SMI312 and MAP2 stain the cell body next to axons and dendrites, respectively. By masking the SMI312 staining with the MAP2 staining the overlap is considered the cell body.
IdentifyTertiaryObjects	Removes the cell body from the ‘axons+cell body’ identified before so that only the axons will remain.
IdentifyTertiaryObjects	Removes the cell body from the ‘dendrites+cell body’ identified before so that only the dendrites will remain.
MeasureObjectSizeShape	Measures the size and shape of axons and dendrites.
ConvertObjectsToImage	The skeleton module requires a binary picture, so this module converts the identified axons to a binary image.
ConvertObjectsToImage	The skeleton module requires a binary picture, so this module converts the identified dendrites to a binary image.
MeasureObjectSkeleton	Measures the skeletal features (branches, endpoints, trunks) for the axons starting from the cell body.
MeasureObjectSkeleton	Measures the skeletal features (branches, endpoints, trunks) for the dendrites starting from the cell body.
ExportToSpreadsheet	Exports the data (number of objects, measurements, etc.).