The study of neurite dynamics is critical to the investigation of neural injury and regeneration, neuropathological disorders, neuronal differentiation, and embryonic development.
Measurements of neurite dynamics are commonly utilized as a screening assay in neurotherapeutic drug discovery, and variations in neurite length and branching can forecast neurotoxicity and neuroprotective effects influenced by a compound.
The main method for studying neurite dynamics has been to concatenate measurements of neurite length and branch points taken at single points in time. This method entails growing neuronal cultures, giving them a treatment condition, and stopping the experiment with fixation and immunostaining steps.
Once this has been completed, the acquisition of images and further analysis can be undertaken. Even though this method has been a necessary tool in neurobiology, the process is labor intensive and provides only a single time point of data, which may not reveal the dynamic effects of the treatment.
To solve these challenges, the NeuroTrack assay was created. NeuroTrack software, paired with the IncuCyte ZOOMTM live-content imaging platform, analyzes living cells’ neurite dynamics without the need for a fluorescent label.
The IncuCyte ZOOM captures non-perturbing phase-contrast images of neuronal cultures under physiological conditions over a long amount of time. NeuroTrack software provides data on neurite dynamics by measuring these images.
Users can also collect data from their culture for a complete time course which is beneficial by potentiating a complete understanding of the kinetic effects of the treatment of interest.
NeuroTrack is suitable to use with a variety of neuronal model systems; among others, the software is able to quantify the highly intricate neurites of primary and iPSC-derived cells, and the hardy processes of differentiated Neuro-2a and PC-12 cells.
Preparation of Axol Cortical Neural Stem Cells for NeuroTrack Assay
Axol Cortical Neural Stem Cells can be plated, thawed and differentiated on the first day of the NeuroTrack assay. On the other hand, they can be expanded before differentiation, or they can be dissociated and re-plated after differentiation to adapt to the needs of the experiment.
For instructions on thawing, passaging and differentiation, please read Axol’s ‘Culture and Differentiation of Axol hNPCs’ instruction manual.
Cell Seeding to a 96-well plate (NeuroTrack Assay)
- Thaw the Axol Sure Bond coating solution overnight at 4 °C.
- Dilute the Axol Sure Bond stock solution (50X) in WFI-grade water to create working solution, for example 120 μL in 6 mL.
- Coat the surface well with 100 μL Axol Sure Bond working solution.
- Incubate the culture vessel overnight at 37 °C.
- Remove and rinse wells once with PBS quickly before the cells need plating.
- For a 96-well assay, adjust density to 100K cells per ml in 1:1000 Axol Sure Boost supplemented media and plate 100 μl per well (10K cells per well) in the coated 96-well TPP plate.
- Leave at RT for 30 minutes to minimize edge effects.
- Place plate in IncuCyte ZOOM and observe the neurite dynamics for the rest of the experiment.
- 24 hours after seeding, remove the neural enhance supplemented media and replace with pre-warmed 1:1000 Axol Neural Advance supplemented media.
- Two days after differentiation, replace 50% of the media (for example 50 μl) with 2x FAC Axol Neural Advance (1:500) for an additional two to three days.
- After this time carry out a 100% media replacement with Complete Axol Neural Maintenance Medium. Once completed, treatments may be added if necessary.
- Replace 50% of media every two to three days.
Raw data can be exported to Excel (Neurite length (mm/mm2, Branch points (1/mm2) and normalization to cell body cluster) and re-arranged according to treatments. Utilize the GraphPad Prism 5.0 to track changes in the analyzed parameter as a function of time (hours). Obtain the area under the curve from this time-course data.
Utilize Excel to plot the log of molar concentration of test compound against the impact of each compound on the area under the curve. Carry out a nonlinear regression, using the 4 parameter logistic model (fitting model 204), to provide estimates of compound pIC50 values, Hill coefficient and minimum and maximum values (XLfit Version 220.127.116.11.).
About AXOL Biosciences
Axol specializes in human cell culture.
Axol produces high quality human cell products and critical reagents such as media and growth supplements. We have a passion for great science, delivering epic support and innovating future products to help our customers advance faster in their research.
Our expertise includes reprogramming cells to iPSCs and then differentiating to various cell types. We supply differentiated cells derived from healthy donors and patients of specific disease backgrounds. As a service, we also take cells provided by customers (primary or iPSC) and then do the reprogramming (when necessary) and differentiation. Clearly, by offloading the burden of generating cells, your time is freed up to focus on the research. Axol holds the necessary licenses that are required to do iPSC work.
The package wouldn't be complete without optimized media, coating solutions and other reagents. Our in-house R&D team works hard to improve on existing media and reagents as well as innovate new products for human cell culture. We also supply a growing range of human primary cells; making Axol your first port of call for your human cell culture needs.
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