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The TUNEL assay marks cells whose DNA has been cut into fragments, a feature of late-stage apoptosis. The enzyme terminal deoxynucleotidyl transferase (TdT) attaches a labelled nucleotide to the exposed 3′-OH ends of broken DNA, and the label is then read by microscopy, flow cytometry or brightfield staining. A positive signal shows DNA strand breaks, not apoptosis by itself, so it is normally paired with a second method.
Definition and scope
TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) is a labelling method for DNA damage in fixed cells and tissue. Thermo Fisher’s TUNEL assay documentation explains that late-stage apoptosis fragments DNA, and that TUNEL labels the exposed 3′-OH ends of those breaks. Elmore’s review of apoptosis in Toxicologic Pathology (2007) describes TUNEL as identifying cells with DNA strand breaks in situ, which makes it a way to assay the endonuclease cleavage products of apoptosis.
“TUNEL staining” is the same assay described by its result: cells or tissue sections that carry the label are said to be TUNEL-positive. It is a DNA-damage readout in fixed samples. It does not measure the early apoptotic membrane changes that Annexin V detects (see Annexin V/PI apoptosis assay setup for flow cytometry), and it does not count viable cells (see hemocytometer cell counting and viability).
How the TUNEL assay is used in practice
According to Thermo Fisher’s documentation, TdT adds a modified dUTP to the 3′-OH ends of fragmented DNA. The modified nucleotide is either detected directly or becomes a handle for a second detection step. Samples must be fixed and permeabilized so the enzyme and detection reagents can reach the nucleus. Results are expressed as the percentage of positive cells, calculated against a negative control, and DNase I-treated tissue is shown as a way to generate a deliberate positive TUNEL signal.
Elmore (2007) calls the method very sensitive, able to detect even a single cell by fluorescence microscopy, and lists cost as a disadvantage. The same review notes that the exact number of strand breaks required for detection is not known.
Label chemistries and readouts compared
The table compares the detection chemistries described in Thermo Fisher’s TUNEL assay documentation. It describes what the manufacturer states, not how the products perform in your system.
| Chemistry | How the signal is generated | Readout and sample type stated |
|---|---|---|
| BrdUTP (APO-BrdU kit) | BrdU incorporated by TdT, detected with an Alexa Fluor 488 anti-BrdU antibody | Optimized for flow cytometry of cultured cells; includes propidium iodide for total DNA content and fixed positive and negative control cells |
| EdUTP with click chemistry (Click-iT Alexa Fluor) | Copper-catalysed click reaction attaches a dye azide to the incorporated EdUTP | High-content screening and microscopy of cultured cells; assay time about 2 hours |
| EdUTP, lower-copper click (Click-iT Plus) | Click reaction with copper concentration optimized for compatibility | Preserves fluorescent protein signal and phalloidin binding; optimized for tissue sections |
| EdUTP, colorimetric (Click-iT Colorimetric) | Biotin azide, then streptavidin-peroxidase and DAB | Brown signal read by brightfield microscopy |
| Directly labelled nucleotides (for example fluorescein-dUTP, biotin-dUTP) | One-step labelling by TdT | Listed by Thermo Fisher as label options; it states the two-step click method detects a higher percentage of apoptotic cells than one-step labelled nucleotides |
The page states that the original Click-iT chemistry uses a high copper concentration and is not recommended with fluorescent proteins, which is why the Click-iT Plus version was optimized for them. If a sample expresses a fluorescent protein or is stained with phalloidin, that is the compatibility point to check first.
Where it matters for labs and supply decisions
Because TUNEL needs fixed material and several reagent classes, a kit checklist is more useful than a product name. When choosing a kit, check:
- Sample type: the manufacturer distinguishes cultured-cell kits (flow cytometry, high-content imaging) from a tissue-section kit and a brightfield kit.
- Detection instrument: flow cytometer, fluorescence microscope or brightfield microscope, matching the chemistry in the table.
- Fixation and permeabilization: required for the assay, so confirm these reagents are in the kit or are supplied separately.
- Controls: a negative control to calculate percent positive; DNase I-treated tissue is shown by the manufacturer as a way to generate a deliberate positive signal. Some kits include fixed positive and negative control cells.
- Co-stains: fluorescent proteins and phalloidin constrain the click chemistry; some kits add propidium iodide for DNA content.
For the imaging side of the same workflow, see immunofluorescence protocol, controls and troubleshooting and immunohistochemistry protocol, controls and troubleshooting.
What a TUNEL-positive cell does and does not show
It shows
- The cell contains DNA strand breaks with accessible 3′-OH ends (Thermo Fisher; Elmore 2007).
It does not show on its own
- That the cell died by apoptosis. Elmore (2007) states the method is subject to false positives from necrotic cells and from cells in the process of DNA repair, and gene transcription can also contribute.
- A meaningful percentage without a negative control: the manufacturer calculates percent positive against one.
Elmore states TUNEL should be paired with another assay to confirm that cell death is apoptotic.
Common misunderstandings
- “TUNEL-positive means apoptotic.” It means DNA breaks were labelled. Necrotic cells and cells repairing DNA can also label (Elmore 2007).
- “TUNEL detects early apoptosis.” The manufacturer describes DNA fragmentation as a late-stage event.
- “Any TUNEL kit works on any sample.” The chemistry determines the readout and sample fit, as the table shows.
FAQ
What does a TUNEL assay detect?
It detects DNA strand breaks by labelling exposed 3′-OH ends with a TdT-added modified nucleotide, a feature of late-stage apoptosis.
What is TUNEL staining?
TUNEL staining is the TUNEL assay as seen in samples: labelled nuclei in fixed cells or tissue sections are TUNEL-positive, read as fluorescence or, with a colorimetric kit, a brown DAB signal.
Can TUNEL give false positives?
Yes. Elmore (2007) reports false positives from necrotic cells and cells undergoing DNA repair, and states an additional method should be used to confirm apoptosis.
Which controls does it need?
A negative control, against which percent positive is calculated, and a sample deliberately damaged with DNase I to generate a positive signal (Thermo Fisher). Some kits supply fixed positive and negative control cells.
Sources
- Thermo Fisher Scientific, TUNEL assays (manufacturer documentation), checked 2026-10-29.
- Elmore S. Apoptosis: a review of programmed cell death. Toxicologic Pathology, 2007, checked 2026-10-29.
