Page 107 - Live-cellanalysis handbook

Kinetic Neurite Analysis Assays

A Neurite length B Neurite length
(mm/mm ) (mm/mm ) 2
2
200 200
150 333 µM 150
111 µM IC 29 µM
50
37 µM
100 12 µM 100
4 µM
1.4 µM
50 Vehicle 50
0 0
0 100 200 300 -7 -6 -5 -4 -3 -2
Time (h) Time (h)
C Neurite length D Neurite length
(mm/mm ) (mm/mm )
2
2
Figure 4. Glutamate-induced retraction of 250 200
neurite projections. Time-course of effects of
glutamate addition at day 9 (arrow) on neurite 200 Vehicle
length is shown in A (mean + SEM, n=4). (B) 150 IC 0.44 µM
50
Concentration-response analysis for data in A 150 250 µM Glutamate
+ 10 nM MK801
(mean + SEM, n=4) taken at 286 h time-point. (C) + 4.6 nM MK801 100
Addition of increasing concentrations of MK-801 100 + 1.5 nM MK801
10 minutes prior to addition of 250 μM glutamate 50 + 57 pM MK801 50
(arrow) protects neurites from glutamate toxicity
(mean + SEM, n=4). (D) Concentration-response 0 0
data for MK-801 effects measured at 298 h (mean 0 100 200 300 -12 -10 -8 -6 -4
+ SEM, n=4). Time (h) Time (h)

Live-cell analysis utility for characterization and development of iPSC-derived neuronal models
Neurite analysis can also be used to characterize and optimize the coatings, with dramatic cell clustering and radial neurite
basic culture conditions when plating and maintaining iPSC. An cabling demonstrated with PDL and PLO, while the morphology
example of this is shown in Figure 5 where the culture of iCell® of the monolayer on PEI plates were more homogenous and
neurons (CDI) were tested in three common culture substrates representative of classical neurite outgrowth. Secondary coating
(PDL, PLO, PEI) w/wo secondary laminin or Matrigel coating. with either laminin or Matrigel produced a neuronal monolayer
iCell Neurons were seeded at 50,000 cells/well on PDL, PLO or with more pronounced neurite outgrowth, and laminin was chosen
PEI +/-Matrigel or +/-laminin, and images were capture at 10X for further experimentation.
magnification. Different morphologies were observed between

PDL PLO PEI

Alone

Matrigel

Laminin

Figure 5. Determination of optimal adherence and morphology of iCell Neuron monocultures seeded on PEI + laminin iCell Neurons were seeded at 50,000 cells/
well on PDL, PLO or PEI +/- Matrigel or +/- laminin. Cells plated on PDL or PLO formed large neurospheres by DIV14 in the presence or absence of additional
laminin or Matrigel coating. Cells plated on PEI +/- laminin or Matrigel displayed a more homogenous monolayer. All images captured at 10x magnification.

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