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PLoS One. 2013;8(2):e56690. doi: 10.1371/journal.pone.0056690. Epub 2013 Feb 22.

Label-free detection of neuronal differentiation in cell populations using high-throughput live-cell imaging of PC12 cells.

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1
Freiburg Institute for Advanced Studies, Albert-Ludwigs-University Freiburg, Freiburg, Germany.

Abstract

Detection of neuronal cell differentiation is essential to study cell fate decisions under various stimuli and/or environmental conditions. Many tools exist that quantify differentiation by neurite length measurements of single cells. However, quantification of differentiation in whole cell populations remains elusive so far. Because such populations can consist of both proliferating and differentiating cells, the task to assess the overall differentiation status is not trivial and requires a high-throughput, fully automated approach to analyze sufficient data for a statistically significant discrimination to determine cell differentiation. We address the problem of detecting differentiation in a mixed population of proliferating and differentiating cells over time by supervised classification. Using nerve growth factor induced differentiation of PC12 cells, we monitor the changes in cell morphology over 6 days by phase-contrast live-cell imaging. For general applicability, the classification procedure starts out with many features to identify those that maximize discrimination of differentiated and undifferentiated cells and to eliminate features sensitive to systematic measurement artifacts. The resulting image analysis determines the optimal post treatment day for training and achieves a near perfect classification of differentiation, which we confirmed in technically and biologically independent as well as differently designed experiments. Our approach allows to monitor neuronal cell populations repeatedly over days without any interference. It requires only an initial calibration and training step and is thereafter capable to discriminate further experiments. In conclusion, this enables long-term, large-scale studies of cell populations with minimized costs and efforts for detecting effects of external manipulation of neuronal cell differentiation.

PMID:
23451069
PMCID:
PMC3579923
DOI:
10.1371/journal.pone.0056690
[Indexed for MEDLINE]
Free PMC Article
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