Page 104 - Live-cellanalysis handbook

Live-Cell Analysis Handbook — Third Edition

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IncuCyte Neurite Analysis Assays at a glance
In order to measure neurite dynamics, the IncuCyte® Live-Cell When studying neurite retraction, or in cases where co-cultures are
Analysis System automatically acquires and analyzes images over required, neuronal fluorescent labeling methods can be employed.
days or weeks in 96- or 384-well formats. Image acquisition is In these cases, phase-contrast images alone may not able to
achieved without the motion of a motorized stage that can be discriminate the neuronal projections from either neurite debris or
disruptive to sensitive cells; instead, the optical train moves while support cells. In order to identify neuronal processes, neurons are
cells stay stationary. In addition, cells are not subject to loss of transduced in a single step protocol using IncuCyte® NeuroLight
environmental control at any point in the experiment. Post-image Lentivirus Reagent. This VSV-G pseudotyped lentivirus encodes
acquisition, the IncuCyte® NeuroTrack Analysis Software Module a non-perturbing fluorescent protein driven off of a synapsin
is used to segment neuronal cell bodies and neurites and quantify promoter to strengthen neuronal expression and minimize non-
biologically relevant processes such as neurite initiation, neurite neuronal crossover. Phototoxicity associated with repeat exposure
extension, branching, and loss of neurite length due to retraction. to short wavelength lights must also be avoided when analyzing
Either label-free or fluorescent methods may be employed sensitive neuronal models, so longer wavelength fluorophores (e.g.
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depending on the cell culture model utilized and the scientific orange, red) are preferred. Once fluorescent images are acquired,
question at hand. NeuroTrack algorithms are employed to quantitatively assess neurite
morphology and reveal temporal information.
For studies of neurite outgrowth and branching in simple mono-
culture systems, neurite parameters are derived ‘label-free’ Both label-free and fluorescent IncuCyte Neurite Analysis assays
using phase-contrast images. Cell bodies are segmented from can be combined with apoptosis measurements to gain further
background based on texture and/or brightness, and neurites insight into neurotoxic effects. Multiplexing apoptosis measured
(linear features) are segmented based on width and brightness. By using IncuCyte® Annexin NIR and neurite measurements enables
normalizing the neurite length to the number of cell bodies, it is real-time tracking of neuronal morphology and cell health within
possible to directly compare the rates of outgrowth. the same population of cells in every single well.

Shortcomings of Traditional Assays Live-Cell Imaging and Analysis Approaches

• Data obtained from a single, pre-defined time point yields • Continuous, real-time data can be collected over days, weeks,
minimal dynamic insight. or months without loss of environmental control or physical
movement of cultures.
• Concatenated end point experiments are subject to cell • The same network of cells is repeatedly interrogated over time,
seeding artifacts and require large cell numbers. yielding maximum information from precious cells.
• Fixation and immunostaining steps are labor-intensive and • Neurites are identified without fixation and multiple wash
destroy delicate neurites. steps. Label-free and non-perturbing fluorescent approaches
are available.
• Co-cultures cannot be studied as the entire population is • Purpose built software tools and guided interface enables
analyzed indiscriminately. non-expert operators to perform image processing and
generate publication-ready graphics.

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