Neurite Outgrowth Assay

Assessment of Neurite Outgrowth Using a High Content Screening Platform

The neurite outgrowth assay uses human iPSC-derived neurons (other cell types available upon request).
Cyprotex’s neurite outgrowth assay uses high content screening (HCS) technology to monitor neurite outgrowth.
Neurons are plated on laminin-coated 384-well plates 1 hour prior to treatment. After treatment with test articles and control compounds, neurons are maintained in a humidified environment at 37°C with 5% CO₂ for 72 hrs (optimal). At the end of the treatment period, cells are fixed, permeabilized and stained for evaluation of neurite outgrowth and cell health.
This assay provides a viable in vitro system for assessing compounds that interfere or promote normal neurite outgrowth in neurons, providing a platform for preclinical drug safety, drug discovery and disease modelling.

Neurite outgrowth is a requisite for an accurate functional network of neurons during development. It is also crucial for neuronal plasticity, as well as neuronal regeneration.

¹Salto R et al., (2015) PLoS One 10(8); e0135614

Protocol

Neurite Outgrowth Assay Protocol

Cell Type	Human iPSC-derived neurons (other cell types available upon request)
Analysis Platform	ArrayScan VTi or CellInsight CX7 (Thermo Scientific)
Test Article Concentrations	10 point dose-response curves in triplicate (dependent on customer requirements)
Quality Controls	Negative controls: 0.2% DMSO (vehicle) and chlorpromazine Positive control: nocodazole
Data Delivery	This assay has been optimized to assess cell health and neurite outgrowth utilizing a neuronal profiling bioapplication. Valid cell count, mean neurite average length and neurite total length per neuron are reported.

Data

Data from Cyprotex's Neurite Outgrowth Assay

	EC₅₀ (µM)
Valid neuron count	> 1
Mean neurite average length	0.04933
Neurite total length per neuron	0.03191

Figure 1
Evaluation of cell health and neurite outgrowth for positive control nocodazole tested in human iPSC-derived neurons.

Positive control nocodazole causes a significant decrease in mean neurite average length and neurite total length per neuron over the range of concentrations tested. Nocodazole did not have significant effects on cell viability at these concentrations.

Figure 2
High content images of human iPSC-derived neurons after 72 hr treatment with nocodazole over a range of concentrations.

Images show a decrease in neurite outgrowth in a dose response manner while valid cell count is not significantly affected.

	EC₅₀ (µM)
Valid neuron count	8.645
Mean neurite average length	8.950
Neurite total length per neuron	8.383

Figure 3
Evaluation of cell health and neurite outgrowth for negative control chlorpromazine tested in human iPSC-derived neurons.

Negative control chlorpromazine causes a significant decrease in mean neurite average length, neurite total length per neuron and valid neuron count over the range of concentrations tested. There is a direct correlation between cell loss and neurite length.

Figure 4
High content images of human iPSC-derived neurons after 72 hr treatment with 100 and 0.2µM chlorpromazine. At 100µM, chlorpromazine causes cell death. At the lowest concentration of 0.2µM, cell loss and neurite outgrowth are unaffected.