TUNEL assay

TUNEL Assay

TUNEL assay showing apoptotic cells stained in tissue.

The TUNEL assay (Terminal deoxynucleotidyl transferase dUTP nick end labeling) is a method used in molecular biology to detect apoptosis by labeling DNA strand breaks. This technique is widely used in research to identify and quantify apoptotic cells in a population.

Principle

The TUNEL assay is based on the ability of the enzyme terminal deoxynucleotidyl transferase (TdT) to label the 3'-hydroxyl ends of DNA fragments with modified nucleotides. During apoptosis, DNA is cleaved into oligonucleosomal fragments, which can be detected by the incorporation of labeled nucleotides at the break sites.

Procedure

The procedure involves several key steps:

1. Fixation and Permeabilization: Cells or tissue sections are fixed to preserve cellular structures and permeabilized to allow access of the TdT enzyme to the DNA.
2. Labeling Reaction: TdT catalyzes the addition of labeled dUTP to the 3'-OH ends of DNA fragments. The label can be a fluorescent dye or a biotin tag, which can be detected using appropriate methods.
3. Detection: The labeled DNA is visualized using fluorescence microscopy or other detection systems, allowing for the identification of apoptotic cells.

Applications

The TUNEL assay is used in various fields of biological research, including:

• Cancer Research: To study the effects of anti-cancer drugs on inducing apoptosis in tumor cells.
• Neuroscience: To investigate neuronal cell death in neurodegenerative diseases.
• Developmental Biology: To examine programmed cell death during embryonic development.

Advantages and Limitations

The TUNEL assay is a sensitive method for detecting apoptosis, but it has some limitations:

• Advantages:

 * High sensitivity for detecting DNA fragmentation.
 * Can be used on a variety of sample types, including tissue sections and cell cultures.

• Limitations:

 * May produce false positives due to DNA damage not related to apoptosis.
 * Requires careful optimization of assay conditions to avoid non-specific labeling.

Related Pages

• Apoptosis
• DNA fragmentation
• Terminal deoxynucleotidyl transferase
• Fluorescence microscopy