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Bivariate flow cytometry DNA/BrdUrd analysis of plant cell cycle
S Lucretti1, L Nardi, P T Nisini
1ENEA, C.R. Casaccia, Department INN, Rome, Italy. LUCRETTI@casaccia.enea.it
Summary
This study presents a flow cytometry protocol for plant cell cycle analysis using DNA content and bromodeoxyuridine (BrdUrd) staining. Optimized conditions ensure accurate cell cycle phase determination in plant root tip cells.
Area of Science:
- Plant biology
- Cell biology
- Molecular biology
Background:
- Accurate cell cycle analysis is crucial for understanding plant growth and development.
- Flow cytometry offers a quantitative method for assessing cell cycle progression.
- Standardized protocols are needed for reliable DNA content and DNA synthesis analysis in plants.
Purpose of the Study:
- To develop and optimize a flow cytometry protocol for plant cell cycle analysis.
- To enable simultaneous quantification of nuclear DNA content and bromodeoxyuridine (BrdUrd) incorporation.
- To establish robust methods for analyzing root tip cells from Vicia faba, Pisum sativum, and Zea mays.
Main Methods:
- Indirect immunofluorescence staining for BrdUrd incorporation.
- Propidium iodide staining for nuclear DNA content quantification.
- Optimization of partial DNA denaturation (HCl concentration and time), BrdUrd concentration, and incorporation time.
Main Results:
- Identified optimal conditions: 10 µM BrdUrd for 30 min pulse and 1.5 N HCl for 30 min at 25°C for DNA denaturation.
- Achieved DNA content histograms with a coefficient of variation < 4%.
- Developed a protocol using formaldehyde-fixed isolated nuclei to prevent non-specific staining.
Conclusions:
- The described flow cytometry protocol provides a reliable method for plant cell cycle analysis.
- Optimized conditions ensure high-quality data with minimal variability.
- The protocol's stability allows for long-term storage of fixed nuclei prior to analysis.