Page 108 - Live-cellanalysis handbook

Live-Cell Analysis Handbook — Third Edition

Once culture conditions were optimized for iCell Neurons in assessed with Fluorescent NeuroTrack software (Figure 6), which
monoculture on PEI + laminin, they were subsequently tested to demonstrated robust neurite outgrowth. Although iCell Neurons in
evaluate if the conditions translated to iCell Neurons plated in co- co-culture with astrocytes did not display a dramatic difference in
culture with primary rat astrocytes. ICell Neurons and primary rat morphology or neurite length with other coating conditions (data
astrocytes were seeded at 10,000 and 15,000 cell/well respectively. not shown), we recommend seeding on PEI + laminin to maintain
The ICell neurons were then infected with IncuCyte® NeuroLight consistency with monoculture experiments
Red, images captured at 20X magnification, and neurite length was

PEI/laminin
DIV 6

PEI/laminin
DIV 14

PEI + laminin mean neurite length
Neurite length [RED]
(mm/mm ) 2
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0 Figure 6. PEI + laminin coating enables robust neurite outgrowth in iCell
-10 Neuron co-culture with primary rat astrocytes. Cell culture plates were coated
0 1 2 3 4 5 6 7 8 9 10 11 12 13 with PEI + laminin. All images captured at 20X magnification. Each data
Time (days) point represents mean =/-SEM, n=4.

Multiplexed, kinetic measurements of neurite dynamics and cell health
The flexibility of the IncuCyte system, integrated software, and (IGluta Neuron). Addition of both glutamate and kainate at DIV14
associated non-perturbing reagents enables users to multiplex produced a concentration- and time-dependent decrease in
kinetic measurements of neurite length and branch points with neurite length with concomitant increase in cell death in iGluta
cell health readouts in one assay platform, which spares precious Neurons in co-culture with primary rat astrocytes. To investigate
sample, augments standardization, and enhances throughput for a potential mechanism mediating this toxicity, AMPA receptor and
both mono-culture and co-culture workflows. Figure 7 shows NMDA receptor antagonists (NBQX and MK801, respectively) were
the use of fluorescent (co-culture) NeuroTrack analysis software added at DIV14 along with glutamate or kainate. NBQX and MK801
multiplexed with IncuCyte Annexin V NIR apoptosis reagent reduced the neurotoxic effects of both glutamate and kainate,
to quantitatively characterize potential long term neurotoxic suggesting that these effects are, at least in part, glutamate
effects of glutamate and kainate in hiPSC-derived neurons receptor-mediated.

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