Page 110 - Live-cellanalysis handbook

Live-Cell Analysis Handbook — Third Edition

Conclusions

IncuCyte live-cell neurite analysis assays enable long-term • Assays are conducted in an optimal environment so that
examination of neurite dynamics in primary, immortalized and iPSC- neurite dynamics can be studied over extended time periods
derived neuronal models. These assays are ideal for both optimizing (days or weeks), ideal for studying effects that may not
in vitro models and performing drug pharmacology studies. By present until later points in time. Cells do not experience loss
acquiring images in a physiologically relevant environment, and of environmental control or physical movement as data is
using robust, integrated software analysis, neurite analysis can be collected.
extended from a traditional ‘fix-and-stain’ approach, to kinetic,
non-invasive evaluation of mono- or co-culture models to detect • All data and time points can be verified by inspecting
pharmacological and genetic manipulations that alter neurite individual images and/or time-lapse movies. Observation of
formation, elongation, and disruption. This approach also allows cell morphology provides additional validation and insight
users to optimize the culture conditions and maintenance of into the biological effect of treatment groups.
iPSC-derived neuronal models as well as providing a method to
analyze cell health by deploying longer wavelength, non-perturbing • Assays are either label-free or employ non-perturbing
fluorophores. The IncuCyte approach for real-time, long-term IncuCyte reagents that can be multiplexed (e.g. to readout
quantitative analysis of neuronal morphology and cell health fills a neurite length and cell death). Cell sparing, lab-tested
critical need in the study of human neurophysiological disorders and protocols are provided to minimize troubleshooting.
iPSC-derived neurons.
• Images are automatically acquired and analyzed in 96- or
• The assay is flexible and can be utilized to study neurite 384-well format using an intuitive user interface. This allows
outgrowth or retraction in a wide range of cell types, from for rapid assay optimization.
large immortalized cell lines to primary neurons and iPSCs, in
mono- or co-culture. • Low well-to-well variability and minimal user effort make the
assay amenable to medium throughput screening.
• Sensitive, kinetic measurements of neurite length,
branch points, and cell body clusters capture a complete • The combination of long-term image-based analysis
description of a highly dynamic process that cannot and microplate throughput in a physiologically relevant
be provided by single time point data alone. Arbitrarily environment enables efficient optimization of culture
defined endpoints are not required. conditions and maintenance of iPSC-derived neuronal models.

References

1. Laissue P. et al. 2017. Assessing phototoxicity in live fluorescence
imaging. Nature Methods; 14: 657–661.

Representative IncuCyte Publications

1. Bressan RB, et al. Efficient CRISPR/Cas9-assisted gene targeting 6. Li S, et al. Decoding the synaptic dysfunction of bioactive human
enables rapid and precise genetic manipulation of mammalian AD brain soluble Aβ to inspire novel therapeutic avenues for
neural stem cells. Development, 2017, Feb 15; 144(4):635-648. Alzheimer's disease. Acta Neuropathol Commun. 2018 Nov 8;6(1):121.

2. Cavaliere F, et al. In vitro a-synuclein neurotoxicity and spreading 7. Muñoz SS, et al. The serine protease HtrA1 contributes to the
among neurons and astrocytes using Lewy body extracts from formation of an extracellular 25-kDa apolipoprotein E fragment
Parkinson disease brains. Neurobiol Dis., 2017, Jul; 103:101-112. that stimulates neuritogenesis. J Biol Chem., 2018, Mar 16;
293(11):4071-4084.
3. Hong W, et al. Diffusible, highly bioactive oligomers represent a
critical minority of soluble Aβ in Alzheimer's disease brain. Acta 8. Robinson M, Douglas S, and Michelle Willerth S. Mechanically stable
Neuropathol., 2018, Jul; 136(1):19-40. fibrin scaffolds promote viability and induce neurite outgrowth in
neural aggregates derived from human induced pluripotent stem
4. Jin, M, et al. An in vitro paradigm to assess potential anti-Aβ cells. Sci Rep., 2017, Jul 24; 7(1):6250
antibodies for Alzheimer’s disease. Nat Commun. 2018, Jul 11;
9(1):2676. 9. Vadodaria KC, et al. Altered serotonergic circuitry in SSRI-resistant
major depressive disorder patient-derived neurons. Mol Psychiatry.
5. Kobayashi W, et al. Culture systems of dissociated mouse and 2019 Jun;24(6):808-818.
human pluripotent stem cell-derived retinal ganglion cells
purified by two-step immunopanning. Invest Ophthalmol Vis Sci.
2018 Feb 1;59(2):776-787.

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