Cell proliferation method: click chemistry based on BrdU coupling for multiplex antibody staining.

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• 5-bromo-2 eal-deoxyuridine (BrdU) incorporation assays are standard for cell cycle analysis.
• Traditional BrdU detection requires DNA denaturation, which destroys protein epitopes, limiting multiplex analysis.
• This limitation hinders simultaneous assessment of DNA synthesis and protein expression.

Purpose of the Study:

• To develop a novel assay for detecting DNA synthesis without DNA denaturation.
• To enable sensitive detection of S-phase cells and preserve protein epitopes for multiplex analysis.
• To improve upon existing methods for cell proliferation and multiplex immunocytochemistry.

Main Methods:

• Utilized 5-ethynyl-2 eal-deoxyuridine (EdU) pulse labeling to incorporate an alkyne into newly synthesized DNA.
• Employed copper-catalyzed
• click chemistry
• to couple an azide probe to the ethynyl residue on EdU.
• Detected the resulting DNA-bound bromouracil moieties using anti-BrdU antibodies without DNA denaturation.

Main Results:

• Successfully detected DNA incorporation of EdU without requiring DNA denaturation.
• The novel method demonstrated higher sensitivity compared to traditional BrdU assays.
• Enabled multicolor and multiplex analysis in flow cytometry (FCM) and imaging.

Conclusions:

• The EdU click chemistry assay provides a sensitive and versatile alternative to BrdU detection.
• This method preserves protein epitopes, facilitating multiplex analysis of DNA synthesis and protein expression.
• It is advantageous for cell cycle studies, particularly when combined with immunocytochemistry.