To validate whether the device can be used for morphometric imaging analyses, we investigated whether we could recapitulate the synaptogenic function of neuroligin-3.31 In the left well compartment, wild-type excitatory neurons were seeded. In the right well compartment, two different excitatory neuronal populations were seeded: wild-type excitatory neurons (no over-expression of NL3) as a control, and neurons with lentiviral infection for wild-type NL3 (overexpression). Images were acquired using the IN Cell Analyzer 6000 (GE Healthcare Life Sciences) with a 40× objective and numerical aperture of 0.6. Three to four wells were used for two batches of culture. Between two and four images were captured as Z-stacks from each side of the well by the IN Cell Analyzer 6000. Images were merged and formatted as maximum intensity projections. Synapse quantification was performed using our previously developed quantification platform Intellicount32 to identify synapsin-positive puncta co-localized with MAP2. Data were expressed as a ratio of right-compartment synapses over left compartment synapses. The left compartment functions as an internal control for the synaptogenic potential of each well (which could vary with cell density, neuronal
