Page 113 - Live-cellanalysis handbook

Kinetic Neuronal Activity Assays

Sample Results

Monitoring neuronal activity via detection of calcium oscillations
IncuCyte NeuroBurst Orange is a neuronal specific, live-cell plated at decreasing cell densities (5-40K/well) in co-culture with
genetically-encoded calcium indicator (GECI) that, when used a fixed number of rat astrocytes (15K per well). As visualized in
along with IncuCyte Live-Cell Analysis System and IncuCyte Figure 1a, fluorescence intensity within the range image strongly
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Neuronal Activity Analysis Software Module, enables automated correlates with cell density, with the highest amount of activity
quantification of transient calcium oscillations of thousands of observed at 40K neurons/well. The range image also provides the
functional neurons within a culture over long periods of time. researcher with a qualitative assessment of morphology, toxicity,
Optimizing cell seeding densities for use with the IncuCyte and transduction efficiency. Summary traces of neuronal activity
Neuroburst Orange reagent for monitoring neuronal activity is provide a quantitative assessment of activity within each well,
illustrated below (Figure 1) using a primary model for neuronal and density of neurons tested was optimal for visualization of
activity; co-culture of primary rat neurons (E18) and primary rat neuronal activity within each scan (Figure 1b) and detection of
astrocytes. In this experiment, E18 rat forebrain neurons were active objects over the full 12-day time course (Figure 1c).

A

B Summary trace activity at Day 12 C Number of active objects over 12 days
Neurons
per well

Figure 1. Optimizing cell seeding densities for use with IncuCyte NeuroBurst Orange for monitoring neuronal activity. Primary rat forebrain neurons
were seeded at 40K (rows A and B), 20K (rows C and D), 10K (rows E and F), and 5K (rows G and H) cells/well. All densities of neurons were plated in
a co-culture with primary rat astrocytes seeded at 15K cells/well and transduced with the NeuroBurst Orange Reagent. (A) 96-well vessel view of the
range image over the course of the scan provides a snapshot of active wells at each time point. (B) Summary traces of fluorescence intensity across
all active objects for the 96-well plate at day 12 provide an overview of activity and display metrics of bursting intensity, active object number and
mean correlation. (C) 96-well throughput with high kinetic reproducibility over 12 days in culture.

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