Flow cytometry in formamide treated cells

Area of Science:

• Cell Biology
• Biotechnology
• Immunology

Background:

• Flow cytometry is crucial for cell analysis.
• Formamide has been used for DNA content measurement in cell cycle studies.
• Extending formamide's utility to multi-parameter analysis is needed.

Purpose of the Study:

• To evaluate formamide's compatibility with simultaneous multi-parameter flow cytometry.
• To adapt formamide-based protocols for DNA content, phenotyping, DNA replication, and fractional DNA content analysis.
• To develop simple, reproducible, and advantageous protocols for these applications.

Main Methods:

• Formamide treatment for sample preparation in flow cytometry.
• Simultaneous detection of DNA content and surface phenotypes.
• Quantification of replicating DNA using click chemistry.
• Measurement of cells with fractional DNA content.

Main Results:

• Formamide treatment is compatible with simultaneous DNA content and surface phenotype analysis.
• Optimal DNA stoichiometric staining (CV < 3, G2/G1 ratio = 2 ± 0.05) and fluorescence maintenance were achieved.
• The procedure improves DNA staining by eliminating paraformaldehyde (PFA) fixation and allows 7-aminoactinomycin D (7-AAD) use.
• Formamide-based permeabilization and fixation effectively resolve sub-G1 cells and ensure high sample recovery (>90%).

Conclusions:

• Formamide treatment offers versatile applications in flow cytometry beyond DNA content analysis.
• Adapted protocols provide simple, reproducible, and advantageous methods for multi-parameter analysis.
• This approach enhances the study of cell cycle, DNA replication, and cell death (necrobiology).