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Image-based quantitation of kainic acid-induced excitotoxicity as a model of neurodegeneration in Human iPSC-Derived Neurons

Excitotoxicity is a feature of many neurodegenerative diseases and acquired forms of neural injury that is characterized by disruption of neuronal morphology. This is typically seen as beading and fragmentation of neurites when exposed to excitotoxins such as the AMPA receptor agonist kainic acid, with the extent to which these occur used to quantitate neurodegeneration. Induced pluripotent stem cells (iPSCs) provide a means to generate human neurons in vitro for mechanistic studies and can thereby be used to investigate how cells respond to excitotoxicity and to identify or test potential neuroprotective agents. To facilitate such studies, we have optimized a protocol for human iPSC differentiation to mature neurons in a 96-well plate format that enables image-based quantitation of changes to neuron morphology when exposed to kainic acid. Our protocol assays neuron morphology across seven excitotoxin concentrations with multiple control conditions and is ideally suited to comparison of neurons generated through differentiation of two isogenic iPSC lines in a single plate. We have included detailed step-by-step protocols for neural stem cell differentiation, neuronal maturation and exposure to kainic acid treatment, as well as different approaches to image-based quantitation that involve immunofluorescence or phase-contrast microscopy.

Funding

FightMND

History

Publication title

Methods in Molecular Biology

ISSN

1064-3745

Department/School

Wicking Dementia Research Education Centre

Publisher

Humana Press

Place of publication

United States

Rights statement

Copyright 2021 Springer Science+Business Media, LLC

Repository Status

  • Restricted

Socio-economic Objectives

Prevention of human diseases and conditions; Expanding knowledge in the biomedical and clinical sciences

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