29: TUNEL assay
Summary
The TUNEL assay labels cells with extensive DNA fragmentation, often used to mark cells undergoing apoptosis.
Also known as
T erminal deoxynucleotide transferase (TdT) d U TP n ick e nd l abeling
Samples needed
Fixed cells or tissue in the format needed for final visualization
Method
TUNEL uses the enzyme terminal deoxynucleotide transferase (TdT) to add labeled dUTP to the 3′ OH of DNA breaks. The label on dUTP depends on the desired visualization method. For instance, for a fluorescence microscopy application, dUTP can be directly attached to a fluorescent molecule. The more DNA breaks there are, the stronger the TUNEL signal will be. TUNEL is used to detect cells with massive amount of DNA damage, especially apoptotic cells, as DNA fragmentation is one step of the apoptotic death program.
To perform the TUNEL assay, samples are first fixed, then solubilized. Solubilizing creates holes in the plasma membrane to allow the reagents to enter the sample. The sample is treated with TdT and labeled dUTP, and visualized appropriately.
If a cell is positive, it has lots of DNA fragmentation.
Controls
Often times, controls for TUNEL staining are not shown in a published work. However, a positive control can be generated by treating a sample with DNAse to cause DNA breakage before staining. A negative control can be performed if a critical assay reagent (i.e. TdT) is left out.
Thumbnail
"Bmp7 functions to promote cell survival in the cloacal endoderm.jpg" ↗ by Xu et al. is licensed under CC BY 4.0 ↗.
Description: Fluorescence image of a TUNEL assay.
Author
Katherine Mattaini, Tufts University