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Phenotyping Cellular Viability by Functional Analysis of Ion Channels: GlyR-Targeted Screening in NT2-N Cells
Katharina Kuenzel1, Sepideh Abolpour Mofrad2, Daniel F Gilbert3
1Institute of Medical Biotechnology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Paul-Gordan-Str. 3, 91052, Erlangen, Germany. Katharina.kuenzel@fau.de.
Abstract:
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.
Insights
This study presents a new method to assess cellular health using glycine receptor (GlyR) function in differentiated stem cells. This functional assay provides a reliable indicator for neurotoxicity testing and central nervous system integrity.
Area of Science:
- Neuroscience
- Cell Biology
- Pharmacology
Background:
- Glycine receptors (GlyRs) are crucial for central nervous system function and are targets for drug development.
- Assessing GlyR function offers insights into cellular viability and neurotoxicity.
- Human pluripotent NTERA-2 (NT2) stem cells differentiate into neurons expressing GlyRs.
Purpose of the Study:
- To develop and describe a protocol for phenotyping cellular viability via functional GlyR analysis.
- To utilize YFP-I152L, a halide-sensitive fluorescent protein, for high-throughput GlyR functional assays.
- To establish a method for assessing the integrity of GlyRs in neuronally differentiated NT2 (NT2-N) cells.
Main Methods:
- Neuronal differentiation of NT2 stem cells using retinoic acid.
- Transient transfection of NT2-N cells with the YFP-I152L reporter.
- High-content imaging for functional analysis of GlyR activity and data interpretation.
Main Results:
- Successful neuronal differentiation of NT2 stem cells.
- Expression of functional GlyRs in differentiated NT2-N cells.
- Demonstration of YFP-I152L as a viable reporter for GlyR function in a high-throughput assay.
Conclusions:
- The described protocol enables robust functional analysis of GlyRs in neuronally differentiated stem cells.
- This method serves as a valuable tool for in vitro neurotoxicity and developmental neurotoxicity testing.
- The YFP-I152L reporter facilitates high-throughput assessment of GlyR integrity and cellular viability.
