Analysis of apoptosis by cytometry using TUNEL assay

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Apoptosis is characterized by DNA fragmentation due to endonuclease activation.
• DNA strand breaks are a gold standard for identifying apoptotic cells.
• The TUNEL assay detects DNA strand breaks via fluorochrome-labeling of 3'-OH termini.

Purpose of the Study:

• To describe a sensitive variant of the TUNEL assay for detecting DNA strand breaks.
• To present modifications for using various fluorochrome-tagged nucleotides.
• To enable correlation of apoptosis with cell cycle phase.

Main Methods:

• Utilizing terminal deoxynucleotidyl transferase (TdT) for in situ labeling of DNA strand breaks.
• Employing brominated deoxyuridine triphosphate (Br-dUTP) for labeling, detected with anti-Br-dUAb.
• Implementing concurrent DNA staining (propidium or DAPI) for multiparameter analysis.

Main Results:

• The Br-dU-labeling TUNEL assay demonstrates superior sensitivity in detecting DNA breaks.
• The protocol allows for the use of diverse fluorochrome-tagged deoxynucleotides.
• Flow cytometry or laser scanning cytometry can correlate apoptosis induction with cell cycle phase.

Conclusions:

• The Br-dU-labeling TUNEL assay is a highly sensitive method for apoptosis detection.
• This methodology facilitates the study of apoptosis across different cellular contexts.
• Multiparameter analysis enhances the understanding of apoptosis regulation.