Phenotyping Cellular Viability by Functional Analysis of Ion Channels: GlyR-Targeted Screening in NT2-N Cells
Glycine receptor chloride channels (GlyRs) are attractive drug targets for therapeutic intervention and are also more and more recognized in the context of in vitro neurotoxicity and developmental neurotoxicity testing. Assaying the functional properties of GlyR can serve as an indicator of cellular viability and the integrity of the developing and mature central nervous system. Human pluripotent NTERA-2 (NT2) stem cells undergo neuronal differentiation upon stimulation with retinoic acid and express a large variety of neuronal proteins—including GlyR. YFP-I152L, a halide-sensitive variant of yellow fluorescent protein, allows high-throughput fluorescence-based functional analysis of GlyRs in NT2 cells. Here we describe a protocol for phenotyping of cellular viability by functional analysis of GlyR in neuronally differentiated NT2 (NT2-N) cells using YFP-I152L as a reporter of functional integrity of GlyRs. The protocol describes neuronal differentiation of NT2 stem cells, transient transfection of NT2-N cells with YFP-I152L as well as functional imaging and analysis of data from high-content imaging.
Key wordsHuman pluripotent embryonal teratocarcinoma stem cells NT2 cells NT2-N cells Glycine receptor chloride channel (GlyR) YFP-I152L Cell viability Toxicological screening
The authors gratefully acknowledge funding of the Staedtler Stiftung and Bavarian Equal Opportunities Sponsorship— Förderung von Frauen in Forschung und Lehre (FFL)—Promoting Equal Opportunities for Women in Research and Teaching. Mofrad SA performed the present work in fulfillment of the requirements for obtaining the degree Dr. rer. biol. hum. at the Friedrich- Alexander University of Erlangen-Nürnberg (FAU).
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