A Protocol for In Vitro High-Throughput Chemical Susceptibility Screening in Differentiating NT2 Stem Cells

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1601)

Abstract

The incidence of neurological diseases including learning and developmental disorders has increased in recent years. Concurrently, the number and volume of worldwide registered and traded chemicals have also increased. There is a broad consensus that the developing brain is particularly sensitive to damage by chemicals and that evaluation of chemicals for developmental toxicity or neurotoxicity is critical to human health. Human pluripotent embryonal carcinoma (NTERA-2 or NT2) cells are increasingly considered as a suitable model for in vitro developmental toxicity and neurotoxicity (DT/DNT) studies as they undergo neuronal differentiation upon stimulation with retinoic acid (RA) and allow toxicity assessment at different stages of maturation. Here we describe a protocol for cell fitness screening in differentiating NT2 cells based on the analysis of intracellular ATP levels allowing for the identification of chemicals which are potentially harmful to the developing brain. The described method is suitable to be adapted to low-, medium-, and high-throughput screening and allows multiplexing with other cell fitness indicators. While the presented protocol focuses on cell fitness screening in human pluripotent stem cells it may also be applied to other in vitro models.

Key words

High-throughput screening Cell-based assays Cell viability Developmental toxicity and neurotoxicity DNT Chemical susceptibility Differentiation Neurotoxicity Cell fitness Drug screening Drug discovery Target validation In vitro toxicity screening CellTiter-Glo Luminescent Cell Viability Assay 

Notes

Acknowledgements

The authors gratefully acknowledge funding of the Staedtler Stiftung. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Internal Medicine 5University Medical Center Erlangen, Friedrich-Alexander-Universität Erlangen-NürnbergErlangenGermany
  2. 2.Friedrich-Alexander University (FAU) Erlangen-NürnbergInstitute of Medical BiotechnologyErlangenGermany

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