Neurons create connections via extensions of their cellular body called dendrites or “processes”. This biological phenomena is referred to as neurite outgrowth and is regulated by complex intracellular signaling events1 . Understanding the signaling mechanisms driving neurite outgrowth provides valuable insight for interpreting neurotoxic compound screening data and for elucidating factors influencing neural regeneration.
Neurite outgrowth is assessed by the segmentation and quantification of neuronal processes2. These neuronal processes can be imaged using a fluorescence microscope and quantified with manual tracing and counting when throughput is low. However, for samples in a higher-throughput microplate format, an automated imaging system paired with analysis software is a more efficient solution.
In this application note, we demonstrate the benefits of using the SpectraMax® i3x Multi-Mode Microplate Reader with the SpectraMax® MiniMax™ 300 Imaging Cytometer in combination with MetaMorph® Microscopy Automation and Image Analysis Software for performing automated neurite outgrowth imaging and analysis. Specifically, we present neurite outgrowth data from mouse embryonic dorsal root ganglion cells donated by Thong C. Ma, Ph.D and Dianna E. Willis Ph.D. of the Burke Medical Research Institute, an affiliate of Weill Cornell Medicine.
- SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices cat. #i3x
- SpectraMax MiniMax 300 Imaging Cytometer (Molecular Devices cat. #5024062)
- MetaMorph Microscopy Automation and Image Analysis Software(Molecular Devices cat. #5042477)
- Neurite Outgrowth Application Module(Molecular Devices cat. #40154)
- CHO-K1 cells (ATCC cat. #CCL-61)
- Recombinant Nerve Growth Factor(EMD Millipore cat. #GF307)
- Anti-ßIII-tubulin antibody(Abcam cat. #52623)
- 5-fluorodeoxyuridine(Sigma cat. #F3503-5MG)
- Neurobasal® Medium(ThermoFisher cat. #21103049)
- B27® Supplement(ThermoFisher cat. #17504044)
Mouse embryonic dorsal root ganglion neurons were isolated from E12.5 embryos and initially cultured in Neurobasal Medium supplemented with B27 Supplement, 50 ng/mL nerve growth factor (NGF), and 10 µM 5-fluorodeoxyuridine for seven days. The 5-fluorodeoxyuridine was used to kill off any Schwann cells. The dorsal root ganglion cells were then removed from the plate, dissociated, and cultured on a monolayer of control CHO cells.
After 24 hours, the co-cultures were fixed with 4% paraformaldehyde. The cells were then processed for fluorescence immunocytochemistry with an anti-ßIIItubulin antibody (1:500, rabbit monoclonal) and a green-fluorescent secondary antibody (anti-rabbit). Afterwards, cell cultures were imaged using the MiniMax cytometer using the settings shown in Table 1.
|Optical configuration||SpectraMax MiniMax 300 Imaging Cytometer|
|Well area settings||12 sites|
|Wavelength settings||541 nm (green fluorescence)|
|Image Acquisition settings||541 exposure: 150 ms
541 focus adjustment: 50 µm
Acquired images were imported into MetaMorph software. Images from each section of the well were loaded into a stack and stitched together into a montage using the software’s image processing features. These image montages were then used for subsequent neurite outgrowth analysis.
MetaMorph software’s Neurite Outgrowth image analysis module was used to identify and measure neurite outgrowth based on the settings shown in Figure 1. The software automatically calculates and reports measurements such as number of processes and process length. The measurements were then exported into Microsoft Excel for further analysis and visualization.
High-quality images of GFP-expressing neurons and their outgrowths were acquired with the MiniMax cytometer (Figure 2). MetaMorph software’s builtin stitching feature was used to create the image montages from the original data generated in SoftMax Pro software (Figure 3). Using MetaMorph software’s Neurite Outgrowth module, cell bodies and their outgrowths were identified, and measurements and analysis were reported automatically (Figure 4). Individual neurons could be selected and their neurite outgrowth measurements displayed (Figure 5).
Data were exported into Excel for additional analysis. In this example, we explored the relationship between the number of processes and total outgrowth (total area covered by outgrowth, measured in pixels) (Figure 6). This is just one example of data analysis that can be performed; other types include comparing neuronal populations or between individual neurons.
When used together, the MiniMax cytometer and MetaMorph software allow researchers to image neurons and acquire detailed information about neurite outgrowth. The MiniMax cytometer images fluorescently stained neurons with great clarity, while MetaMorph software’s suite of image processing and analysis tools enable detailed and quantitative analysis of neurite outgrowth. These two products provide a total solution for researchers to elucidate the mechanisms behind neuron differentiation and regeneration and interpret neurotoxic screening results.
- Kiryushko, Darya, Vladimir Berezin, and Elisabeth Bock. “Regulators of neurite outgrowth: role of cell adhesion molecules.” Annals of the New York Academy of Sciences 1014.1 (2004): 140-154.
- Meijering, E., et al. “Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images.” Cytometry Part A 58.2 (2004): 167-176.