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ADME PK

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.

References

1 Salto R et al., (2015). ß-Hydroxy-ß-methylbutyrate (HMB) promotes neurite outgrowth neuro2a cells. PLoS One. 10(8); e0135614

Other Relevant Articles

Radio NM et al, (2008). Assessment of chemical effects on neurite outgrowth in PC12 cells using high content screening. Tox Sci. 105(1); 106-118

Robinette BL et al, (2011). In vitro assessment of developmental neurotoxicity: use of microelectrode arrays to measure functional changes in neuronal network ontogeny. Front Neuroeng. 4(1); 1-9

Blackmore MR et al, (2010). High content screening of cortical neurons identifies novel regulators of axon growth. Mol and Cell Neuro. 44(1); 43-54

Min H et al, (2007). High content screen microscopy analysis of A beta 1-42-induced neurite outgrowth reduction in rat primary cortical neurons: neuroprotective effects of alpha 7 neuronal nicotinic acetylcholine receptor ligands. Brain Research. 1151; 227-235

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