Measuring DNA content by flow cytometry in fission yeast

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• Flow cytometry is a standard technique for assessing DNA content and cell cycle distribution.
• DNA-binding dyes like propidium iodide and Sytox Green allow for stoichiometric DNA measurement.
• Mammalian cell studies have extensively utilized flow cytometry for DNA replication research.

Purpose of the Study:

• To establish and present basic and advanced flow cytometry protocols for DNA replication analysis in fission yeast (S. pombe).
• To enable multicolor analysis of DNA replication, which has historically lagged in S. pombe compared to mammalian cells.
• To provide researchers with reproducible methods for studying DNA replication dynamics.

Main Methods:

• Utilized flow cytometry for DNA content analysis and cell cycle distribution determination.
• Developed protocols for whole cell and nuclear "ghost" preparations for flow cytometry.
• Implemented two-color imaging techniques incorporating Bromodeoxyuridine (BrdU) incorporation for DNA replication analysis.

Main Results:

• Successfully adapted and optimized flow cytometry protocols for S. pombe DNA replication studies.
• Demonstrated the feasibility of multicolor analysis for examining DNA replication in fission yeast.
• Provided detailed methodologies for various sample preparations suitable for advanced flow cytometry.

Conclusions:

• The presented protocols significantly advance the capability for studying DNA replication in S. pombe using flow cytometry.
• Multicolor flow cytometry analysis is now readily achievable for fission yeast researchers.
• These methods will facilitate deeper insights into DNA replication mechanisms and cell cycle regulation in S. pombe.