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Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy
Published on: June 27, 2018
High throughput quantification of mutant huntingtin aggregates.
Emma L Scotter1, Pritika Narayan, Michelle Glass
1Department of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand.
Journal of Neuroscience Methods
|March 28, 2008
Summary
Automated image analysis methods accurately quantify mutant protein aggregates in neurodegenerative disease research. These tools offer rapid, objective alternatives to manual counting, aiding drug discovery for conditions like Huntington
Area of Science:
- Neuroscience
- Biotechnology
- Cell Biology
Background:
- Mutant protein aggregates are key biomarkers in neurodegenerative diseases such as Huntington's disease.
- Traditional quantification methods (manual counting, cell-free assays) lack efficiency and concurrent cell viability analysis.
Purpose of the Study:
- To develop and validate automated, high-throughput image analysis methods for quantifying mutant huntingtin aggregates within cells.
- To provide objective and rapid alternatives to manual quantification for neurodegenerative disease research.
Main Methods:
- Development of four automated image analysis methods using Metamorph software.
- Automation of cell imaging using a Discovery-1 automated fluorescence microscope.
- Comparison of automated methods against manual counting for accuracy and throughput.
Main Results:
- All four automated methods accurately quantified mutant huntingtin aggregates compared to manual counting.
- The PolyQ assay achieved the highest throughput (0.31 s/image).
- The Cell Scoring assay (19.5 s/image) accurately quantified aggregate-forming cells without bias from cell death.
Conclusions:
- Automated image analysis tools offer efficient and objective quantification of mutant protein aggregates in a cellular context.
- These methods facilitate drug discovery efforts for Huntington's disease and other neurodegenerative disorders.
