Page 112 - Live-cellanalysis handbook

Live-Cell Analysis Handbook — Third Edition

Kinetic neuronal activity assays at a glance

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To evaluate long-term neuronal activity measurements, IncuCyte Activity Analysis Software Module captures these short-term
live-cell imaging and analysis employs an end-to-end solution calcium oscillations via high speed movie acquisition (3 fps for
consisting of instrumentation, software, and reagent utilized in a 30-180s) in every well of a 96-well microtiter plate. Acquired
physiologically relevant environment. The IncuCyte NeuroBurst fluorescent IncuCyte movies are then analyzed to quantify the
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Orange Reagent is a genetically encoded calcium indicator that orange fluorescent signal to derive the active neuronal count
can be expressed in a variety of neuronal cell types, including and connectivity over the course of the experiment, enabling
iPSC-derived models, to gain insight into the dynamic changes continuous characterization of developing networks as they
in activity via measurements of calcium oscillations. In order to become functional and mature. This approach provides researchers
acquire these neuronal activity changes, the reagent encodes the opportunity to continuously analyze the same population of
a fluorescent protein that is excited at a higher wavelength cells long-term, from days to weeks to months, in order to gain
to avoid phototoxic damage to the sensitive neurons that are insight into how and when neurons become active and how their
being analyzed. The IncuCyte system and IncuCyte Neuronal activity changes over time.
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Shortcomings of Traditional Assays Live-Cell Imaging and Analysis Approaches
• Data obtained from at a single point in time yields minimal • Long-term, kinetic evaluation shows longitudinal changes in
insight as neuronal networks develop and mature. network activity to determine when neurons become active
and how their activity changes over time.
• Measurement of activity from a limited number of cells or • Burst rate of thousands of cells in every well of a 96-
by using concatenated single time point data causes high well plate is measured with high accuracy via continuous
variability in assay results. interrogation in the same population of cells over days,
weeks, or month.
• Lack of environmental control and physical movement of • Uninterrupted environmental control provided by a tissue
plate during analysis disturbs sensitive neuronal structures culture incubator, coupled with a non-perturbing reagent
and the biology of interest. and automated image acquisition without physical movement
of sample, protects sensitive neuronal structures and
maintains integrity of data.
• Poor/no visualization of cell morphology and limited spatial • Qualitatively monitor cell morphology using HD phase image
resolution does not allow for evaluation of cell health or and quantify functional connectivity of entire network of
neuronal network formation. active objects in each well of a 96-well plate.
• Complex instrumentation and image processing requires • Purpose built software tools and guided interface enables
expert operation and training for data generation and scientific discovery for even first time users.
analysis.

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