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An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry
Published on: May 13, 2019
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An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry
1Biology Department, Luminex Corporation; mrodrigues@luminexcorp.com.
Journal of Visualized Experiments : Jove
|May 28, 2019
Summary
Multispectral Imaging Flow Cytometry (MIFC) offers an automated solution for the in vitro micronucleus (MN) assay, overcoming limitations of manual scoring and conventional methods. This approach enhances accuracy and throughput for genotoxicity and cytotoxicity testing.
Area of Science:
- Toxicology and Pharmacology
- Cell Biology and Genetics
- Biotechnology and Bioinstrumentation
Background:
- The in vitro micronucleus (MN) assay is crucial for assessing genotoxicity and cytotoxicity.
- Manual microscopy scoring of MN assays is time-consuming and prone to inter-scorer variability.
- Existing automated methods like slide-scanning microscopy and flow cytometry have limitations in image visualization and data storage.
Purpose of the Study:
- To develop and validate a fully automated protocol for the in vitro MN assay using Multispectral Imaging Flow Cytometry (MIFC).
- To leverage MIFC's capabilities to overcome the limitations of traditional manual scoring and conventional flow cytometry.
- To enable high-resolution image acquisition, storage, and automated analysis for improved genotoxicity and cytotoxicity assessment.
Main Methods:
- Human lymphoblastoid TK6 cells were treated with genotoxic agents (Mitomycin C, Colchicine) and a non-genotoxic agent (Mannitol).
- Cells were prepared using hypotonic treatment, fixation, and nuclear staining (Hoechst 33342).
- Automated sample processing and high-resolution image acquisition were performed on the MIFC, followed by automated data analysis.
Main Results:
- The automated MIFC protocol successfully identified, categorized, and enumerated key cellular events for MN assay scoring.
- Statistically significant increases in micronucleus (MN) frequency were detected following exposure to Mitomycin C and Colchicine at various cytotoxicity levels.
- No significant increase in MN frequency was observed after exposure to Mannitol, confirming assay specificity.
Conclusions:
- Multispectral Imaging Flow Cytometry (MIFC) provides a robust, automated platform for conducting the in vitro MN assay.
- This method enhances throughput and reduces scorer bias compared to manual microscopy.
- MIFC facilitates accurate genotoxicity and cytotoxicity evaluation with high-resolution image data storage.
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