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An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry
Published on: May 13, 2019
Fluorescent dye-based simple staining for in vivo micronucleus test with flow cytometer.
Asako Harada1, Kaori Matsuzaki, Akira Takeiri
1Chugai Pharmaceutical Co., Ltd., Gotemba, Shizuoka, Japan.
Mutation Research
|January 8, 2013
Summary
A new fluorescent dye-based flow cytometry method simplifies micronucleus testing. This approach offers a practical, automated way to analyze red blood cells for genotoxicity, improving efficiency in research.
Area of Science:
- Toxicology
- Genetics
- Biotechnology
Background:
- Flow cytometry (FCM) is efficient for micronucleus (MN) testing.
- Current CD71-based FCM uses antibody and dye staining, which can be complex.
- Fluorescent dye-based FCM (FD-FCM) offers a simpler alternative.
Purpose of the Study:
- To develop and validate a practical FD-FCM method for MN testing.
- To assess the utility of FD-FCM for automated scoring of micronucleated immature erythrocytes.
Main Methods:
- Peripheral blood (PB) and bone marrow (BM) erythrocytes from cyclophosphamide-treated rats were used.
- Nucleated cells were removed from BM samples using a cellulose column.
- Erythrocytes were fixed, stained with Hoechst 33258 and propidium iodide (PI), and analyzed by FCM.
Main Results:
- FD-FCM showed comparable results to microscopy for micronucleated immature erythrocytes in PB and BM.
- FD-FCM accurately quantified immature erythrocyte populations in both PB and BM.
- The method demonstrated utility for automated scoring in rat BM and PB MN testing.
Conclusions:
- A simple FD-FCM method involving column purification, fixation, and fluorescent dye staining is effective.
- This method provides a practical approach for automated MN scoring.
- FD-FCM is a valuable tool for genotoxicity assessment in toxicological studies.
