Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Cells undergo apoptosis, or programmed cell death, during both normal development and disease, and the process is the subject of intense research. Naturally, there exists a range of tools to study apoptosis, including both molecular and phenotypic changes.
One established hallmark of apoptosis is DNA fragmentation, commonly measured using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay. The assay uses an enzyme to tag free ends of fragmented DNA with labeled nucleotides, which can be detected using various methods. If you plan to use TUNEL in your own research, here’s what you need to know.
Sample type and detection method
Your method of detection depends in part on the nature of the molecular tag. For DNA labeled with green or red fluorescent molecules, for example, you need a fluorometer, microscope or other fluorescent detection device. If you label cells in suspension, you can use a flow cytometer. Or, if you tag your DNA with biotin and detect with streptavidin-horseradish peroxidase, your signal will be either colorimetric or luminescent, requiring for instance, a luminometer.
Before choosing your detection options, though, you should consider your sample type, says Martin Ramsden, product manager at R&D Systems. Many vendors offer assay kits optimized for just that purpose. R&D Systems, for instance, offers kits for fixed cells, embedded tissues, frozen tissues, suspended cells and cell monolayers, says Ramsden.
Likewise, EMD Millipore's TUNEL assays can be applied to both tissue and whole cells, with peroxidase and fluorescent detection for tissues and fluorescent detection and flow cytometry for whole cells, says product manager Jun Ma.
EMD Millipore’s TUNEL-assay portfolio includes the ApopTag® ISOL Dual Fluorescence Apoptosis Detection Kit for paraffin-embedded tissue, frozen tissue sections, cell suspensions and adherent cells; the Apo-Direct TUNEL Assay Kit for flow cytometry of cells in suspension; and the ApopTag Peroxidase In Situ Apoptosis Detection Kit for staining via peroxidase activity.
Fluorescence and colorimetric detection
The classic nonradioactive TUNEL assay is based on fluorescence—or in a modified form, colorimetric—detection. For example, Promega’s DeadEnd™ Fluorometric TUNEL System is designed for cultured cells and formalin-fixed paraffin-embedded (FFPE) tissue sections. The colorimetric version works with cultured cells but also supports thicker tissue sections.
BioVision also offers a range of fluorescence-based kits for detecting apoptosis in suspended cells, monolayer cells, embedded tissue and frozen tissue. The company’s ApoBrdU™ and ApoDIRECT™ assays detect apoptotic cells using flow cytometry, whereas the ApoBrdU-IHC™ detects DNA fragmentation by dual-color immunohistochemistry (IHC). For GFP-expressing cells, the company recommends its ApoBrdU Red™ kit, which enables dual-color detection of your gene of interest (green) and DNA fragmentation (red) in the same sample, says Poulomi Acharya, a technical specialist at BioVision.
Other vendors also offer fluorescence-based kits, such as Trevigen’s TACS® TdT in situ-fluorescein kit (also distributed by R&D Systems) for fixed, embedded or frozen tissues using fluorescence microscopy, and the FlowTACS® in situ-fluorescein kit for fluorescence microscopy of cells in a monolayer or flow cytometry of cells in suspension.
Trevigen also offers its TACS-XL® assay, which uses a biotin-conjugated BrdU antibody and streptavidin-horseradish peroxidase. “The combination of antibody specificity with the signal-enhancing properties of biotin-streptavidin results in precise cellular labeling and [a high] signal-to-noise ratio,” says Plamena Kirova, marketing manager at Trevigen. For colorimetric detection using 96-well plate readers, there’s Trevigen’s TiterTACS® in situ kit for cells in suspension or in a monolayer.
Kit versatility
Roche Diagnostics offers a line of In Situ Cell Death Detection kits that are designed for flexibility, taking into account that researchers often want to measure apoptosis in a variety of different sample preparations. “Researchers can detect apoptotic cells by fluorescent microscopy, light microscopy or FACS,” says David Guffey, senior technical service consultant at Roche Diagnostics. “Apoptotic cells can be detected in paraffin-embedded or frozen tissue sections, or cells on cover slips or slides.”
Apoptotic cells can even be detected by electron microscopy. Trevigen’s TACS-XL DAB In Situ Apoptosis Detection Kit, for instance, inserts a BrdU nucleotide at DNA ends, which is detected with a biotinylated antibody and streptavidin-conjugated label. Typically, that label is horseradish peroxidase, but it also can be colloidal gold, which can be picked up by EM.
Trevigen also gives researchers the opportunity to optimize different steps of the apoptosis assay for their own particular samples. Or users can select kits optimized for particular cell types, such as Trevigen’s CardioTACS™, NeuroTACS™ II, TumorTACS™, DermaTACS™ and VasoTACS™ kits. “Each kit provides a pre-selected cation, for optimal labeling of free apoptotic DNA ends, and minimized labeling of necrotic cells,” says Kirova.
Potential pitfalls
Although TUNEL-assay kits are well optimized, several pitfalls remain.
One potential hangup is detecting false positives. The single-stranded DNA breaks detected by TUNEL assays can occur in situations other than apoptosis, including necrosis. “If [you are] interested in only apoptosis and concerned about the level of necrosis that is in the tissue or cell sample causing higher than normal false-positive background noise, then [you] should consider TUNEL assays that help distinguish between apoptosis and necrosis better,” says Ma. EMD Millipore’s ApopTag ISOL Dual Fluorescence Apoptosis Detection Kit, for instance, detects only double-stranded DNA breaks. “These breaks are more associated with apoptosis than necrosis, and can be useful especially in diseased tissues that have high levels of necrotic cells,” says Ma.
Another potential pitfall is the timing of your TUNEL assay relative to the start of apoptosis, says Terry Riss, product specialist in cell health at Promega. “Apoptosis is a transient process with appearance and disappearance of markers over time.” Many researchers use two assays that measure apoptosis markers occurring at distinctly different time points to avoid this problem. “The most-often measured endpoint is caspase-3/7 activity, which indicates the execution phase of apoptosis, but morphological observation of apoptotic phenotype is a good orthogonal method,” says Riss.
Lastly, when assaying fixed and permeabilized tissues, pay particular attention to permeability. “One of the potential pitfalls researchers may encounter when using TUNEL-assay kits is the insufficient permeabilization of tissue samples,” says Guffey. Most TUNEL-assay protocols are designed for 10- to 15-µm tissue sections. “If tissue sections are thicker than this, permeabilization times should be extended,” he explains. “Without correct permeabilization, samples will not be labeled and classified as non-apoptotic.”
As apoptosis assays go, TUNEL technology isn't exactly new, dating back to the early 1990s. Yet the assay remains popular, in part because it meshes well with other cytological techniques.
“One benefit of using a TUNEL assay for apoptosis research is the ability to detect surface and intracellular biomarkers [on the same sample],” says Guffey. “As the workflow for TUNEL assays is very similar to and compatible with immunohistochemistry, following the labeling of cells, researchers have the ability to detect other biomarkers using antibodies.”
This convenience, combined with ease of use and reliability, means TUNEL assays are likely to be around for some time to come.
Image: DU145 prostate cancer cells undergoing apoptosis. (Source: Wikipedia)