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A versatile assay for the accurate, time-resolved determination of cellular viability
Toyoki Amano1, Ken ichi Hirasawa, Michael J O'Donohue
1Department of Biology and Geoscience, Faculty of Science, Shizuoka University, 422-8529, Shizuoka, Japan. sbtaman@ipc.shizuoka.ac.jp
Analytical Biochemistry
|March 14, 2003
Summary
A new method accurately measures cell viability using fluorescein diacetate (FDA). This technique allows for precise, time-resolved kinetic analysis of cell death in plant cell cultures.
Area of Science:
- Plant Biology
- Cell Biology
- Biotechnology
Background:
- Accurate determination of cellular viability is crucial for biological research.
- Existing methods may lack precision or time-resolution for kinetic studies.
- Fluorescein diacetate (FDA) is a common indicator of cell viability.
Purpose of the Study:
- To develop a convenient and versatile method for accurate, time-resolved determination of cellular viability.
- To enable kinetic analysis of cell death induction.
Main Methods:
- Utilized fluorescein diacetate (FDA) as a viability probe, measuring fluorescence emission with a spectrofluorimeter.
- Calibrated the assay using viability standards with known proportions of living and dead cells.
- Applied the assay to tobacco BY-2, Chlamydomonas reinhardtii, and Arabidopsis thaliana cell cultures.
Main Results:
- Demonstrated a linear response for cell suspensions within a specific density range (0.5-2.0 x 10^5 cells) using FDA concentrations below 12 µM.
- Achieved a high correlation coefficient (0.998) for tobacco BY-2 cell viability.
- Successfully applied the assay to different plant cell types and performed kinetic analyses of cell death.
Conclusions:
- The developed FDA-based spectrofluorimetric method provides accurate and time-resolved cellular viability assessment.
- This assay is versatile and applicable to various plant cell cultures.
- Enables quantitative analysis of cell death kinetics, including Vmax and LD50 values.