Flow cytometric analysis of chromosomes and cells using a modified BrdU-Hoechst method

Area of Science:

• Cell Biology
• Genetics
• Biotechnology

Background:

• Bromodeoxyuridine (BrdU) incorporation is a common method for labeling newly synthesized DNA.
• Hoechst 33342 is a fluorescent dye that binds to DNA, with its fluorescence quenched by BrdU incorporation.
• Accurate quantification of cell proliferation and cell cycle progression is crucial in various biological studies.

Purpose of the Study:

• To develop and validate a flow cytometry-based method for quantifying DNA synthesis and cell proliferation.
• To establish a technique for distinguishing and measuring successive cell cycles using BrdU and Hoechst staining.
• To assess the temporal dynamics of DNA fluorescence changes during cell division.

Main Methods:

• Chinese hamster cells (B14 F28) were cultured with BrdU for up to four cell cycles.
• Cells and chromosomes were stained with Hoechst 33342 dye for flow cytometry analysis.
• UV exposure was used to reverse BrdU-induced fluorescence quenching, allowing for measurement of total DNA content.

Main Results:

• Chromosomal fluorescence intensity decreased by one step per cell generation due to BrdU incorporation.
• The extent of fluorescence decrease was proportional to the amount of newly synthesized DNA.
• The method successfully established and measured three successive mitoses, demonstrating its utility for cell cycle analysis.

Conclusions:

• The developed method provides a reliable way to measure DNA synthesis and track cell proliferation across multiple generations.
• This technique allows for the determination of the number of cell cycles and cell cycle phase distribution.
• The UV-exposure step is critical for accurate quantification by overcoming BrdU-mediated fluorescence quenching.