TUNEL assay
Summary
Also known as: Terminal deoxynucleotide transferase (TdT) dUTP nick end labeling
Samples needed: Fixed cells or tissue in the format needed for final visualization
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.
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.
Interpretation:
Add more later, but basically if a cell is positive, it has lots of DNA fragmentation.
An investigative analytic procedure
A form of programmed cell death
Deoxyuridine triphosphate, one of the dNTPs added to a growing DNA strand
Preserved in a way that stops all reactions and increases the stability of a sample; fixed samples are no longer living
A substance used in an analytical procedure