In most applications, cell nuclei are identified in the first channel while neuronal cell bodies and neurites are identified in the second channel. Neuronal cell subpopulations can be identified using markers in the third or fourth channels, as shown in Figure 1. Further adding to its flexibility, the ENO BioApplication performs cell-based measurements in up to six imaging channels, and cells are classified on the basis of average intensities. This BioApplication reports a full set of features for each subpopulation, allowing detailed comparison of different cells in the same assay.

In the figures below, the ENO BioApplication was used to detect and measure neurite outgrowth after stimulation of Thermo Scientific Cellomics Neuroscreen^TM-1 cell line with nerve growth factor for 48 hours. The cells were fixed using standard methods and the nuclei were stained with Hoechst dye. Neurons and their neurites were labeled by immunofluorescence using the antibody provided in the Neurite Outgrowth HitKit^TM HCS Reagent Kit that is specific for a protein present in both the neuronal cell body and the neurites. Figure 2 (left panel) shows analysis overlays and images from untreated Neuroscreen-1 cells showing basal levels of neurite outgrowth. In contrast, cells that have been treated with a maximal dose (200 ng/mL) of nerve growth factor exhibit process formation, as shown in the right panel.

The ENO BioApplication successfully distinguishes both conditions, and automatically extracts a number of quantitative features. The graph in Figure 3 illustrates the dose dependent increase in both Branch Point Count and Average Neurite Length as detected by the software, demonstrating the power of this BioApplication for detection of complex changes in neuronal morphology.

Features

• Specifically identifies neuronal cells in a mixed culture and provides objective analysis of neurite outgrowth changes.
  
• Reports several quantitative measures of neuronal morphology for single cells, including neurite length, cell body area, and the number of branch and cross points.
  
• Analyzes and compares subpopulations of neuronal cells identified with different fluorescent markers.
  
• Uses reference wells to automatically determine normal population distributions for a series of measurements and count “responders” to compound treatment.
  
• Validated with both Thermo Scientific Cellomics Neuroscreen^TM-1 cells and primary neuronal cultures.
  
• Highly flexible for user-defined optimization and application across multiple cell types and assays.
  

Benefits

• Automates a labor-intensive assay, providing results in hours instead of weeks.
  
• Enables thorough analyses of the effects of biological and pharmacological agents on neuronal cells at the cell level.
  
• Provides added sophistication to the traditional approach for neuronal analysis.
  
• More objective than manual measurements, with improved reproducibility and reliability.