Measuring DNA content by flow cytometry in fission yeast

Area of Science:

• * Molecular Biology
• * Cell Biology
• * Biotechnology

Background:

• * Flow cytometry is a standard method for analyzing DNA content and cell cycle distribution.
• * Fission yeast is amenable to flow cytometry due to simple sample preparation and dye binding.
• * Multicolor flow analysis of DNA replication in fission yeast remains underexplored compared to mammalian cells.

Purpose of the Study:

• * To present comprehensive protocols for analyzing DNA replication in fission yeast using flow cytometry.
• * To enable advanced studies on DNA synthesis and cell cycle progression in fission yeast.
• * To bridge the gap in multicolor flow analysis for DNA replication in fission yeast.

Main Methods:

• * Development of basic and advanced flow cytometry protocols for fission yeast.
• * Utilizing whole-cell and nuclear "ghost" preparations.
• * Incorporation of two-color imaging with Bromodeoxyuridine (BrdU) and 5-ethynyl-2'-deoxyuridine (EdU) labeling for DNA synthesis estimation.

Main Results:

• * Established robust methods for assessing DNA replication dynamics in fission yeast.
• * Demonstrated the utility of BrdU and EdU incorporation for quantifying DNA synthesis.
• * Provided a framework for multicolor flow analysis of DNA replication in this model organism.

Conclusions:

• * The presented protocols significantly enhance the capability to study DNA replication in fission yeast via flow cytometry.
• * These methods facilitate detailed investigations into cell cycle control and DNA replication fidelity.
• * The study provides essential tools for advancing fission yeast research in molecular and cell biology.