by Caitlin Smith
TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays are a valuable method for detecting DNA fragmentation, which is a hallmark of apoptosis, or programmed cell death. In a TUNEL assay, an enzyme known as terminal deoxynucleotidyl transferase (TdT) identifies nicks, or points of fragmentation, in the sample DNA. TdT catalyzes the addition of dUTP nucleotides that have been labeled previously, for subsequent detection. TUNEL assays come in many flavors, depending on whether you use flow cytometry, light microscopy, fluorescence microscopy and/or microplate assays. Here are some key ideas that are important to consider when choosing the best type of TUNEL assay kit for you.
Consider your sample type
Among your first considerations is which TUNEL assays are best suited to your sample type. Many vendors offer pre-optimized kits for different types of samples. For example, BioVision offers kits suitable for cells; tissues; slides; or frozen, paraffin-embedded tissue sections. “We have the following kits to detect Nuclear Mediated Apoptosis: ApoBrdU™ DNA Fragmentation Assay Kit, ApoBrdU Red™ DNA Fragmentation Kit, ApoBrdU-IHC™ DNA Fragmentation Assay Kit and the ApoDIRECT™ DNA Fragmentation Assay Kit,” says Sunita Gopalan, technical specialist at BioVision. “All kits provide complete components, including positive and negative control cells or slides for convenient detection of DNA fragmentation in cultured cells and tissue sections.”
Specialized, pre-optimized kits may be even more advantageous when working with tissues, because tissue types can vary widely. “Each tissue type has specific challenges associated with it, and the TUNEL assay kit should allow you to perform the assay with minimal need for additional optimization,” says Tom Uveges, senior research and development scientist at Trevigen, whose TUNEL assay kits are distributed by R&D Systems. “By definition, each tissue in the body has unique structural and physical properties related to its specific function. As a result, different tissues require different specific preparative conditions for subsequent biological analysis.” To help researchers do this, Trevigen offers tissue-specific kits containing reagents and protocols optimized for their respective tissue type, such as NeuroTACS™ for neuronal tissue, CardioTACS™ for cardiac tissue, VasoTACS™ for vascular tissue, TumorTACS™ for tumor tissue and DermaTACS™ for dermal tissue. Uveges adds that “if a specialized kit is not available for the tissue of interest, or [if you are] analyzing a compound where there will be low levels of DNA breaks, our TACS XL® kits are recommended.”
Consider permeability and labeling
Two early steps of the assay, the membrane permeability of the reagents and labeling of the nicked DNA, are also important to consider. If you have reason to be concerned about these steps in your particular system, there are various kits that may be helpful. “The TdT enzyme, which is common to all TUNEL assays, is a 60,000 Da protein, the transport of which can be adversely affected by membrane diffusion,” says Brian Almond, senior product manager at Life Technologies. “In order not to compound the problem of membrane permeability, our Click-iT® technology utilizes a smaller size of the detection reagents that enable[s] higher membrane penetration. Compared to an anti-BrdU antibody (MW ~150,000) the Alexa Fluor® azide (MW <~1000) is <1% the size.” He also says the size of the incorporated nucleotide is an important factor. “For a sensitive and reliable TUNEL imaging assay, it is vital that the modified nucleotide is an efficient substrate for TdT. The Click-iT® TUNEL Alexa Fluor® Imaging Assays utilize a dUTP modified with an alkyne that enables the nucleotide to be more readily incorporated by TdT than other modified nucleotides, including BrdUTP, biotin-dUTP or fluorescein-dUTP. Detection is based on the Click reaction, a copper (I) catalyzed reaction between an azide and alkyne.”
BioVision also offers a modified nucleotide for greater effectiveness. “The Apo-BrdU assay uses a two-color TUNEL method for detecting DNA strand breaks in apoptotic cells by flow cytometry,” says Gopalan. “The incorporation of BrdU in the procedure provides greater sensitivity than biotinylated or digoxygenated methods. The Apo-BrdU-Red utilizes the same principle but is designed for GFP-containing cells. The new addition of ApoBrdU-IHC™ provides a complete kit for detecting DNA fragmentation by dual-color immunohistochemistry (IHC). The Apo-DIRECT™ assay offers a simple, one-step labeling of apoptotic cells which can be analyzed by flow cytometry.”
For labeling, Trevigen offers both biotin (TACS® 2 TdT kit) and BrdU (TACS XL® kit) methods. “In experiments where cell proliferation is measured by BrdU incorporation into genomic DNA, apoptotic events can independently be monitored by labeling nicked DNA with biotinylated dUTP,” says Uveges. “The use of BrdU to label nicked DNA is sometimes more sensitive, depending on the level of DNA breaks present in the cell, as a result of higher incorporation of the BrdU nucleotide compared to the biotinylated dUTP.”
Using a TUNEL assay with tissue samples presents special challenges in permeability and labeling. Permeabilization and tissue fixation are critical. “If the tissue is improperly fixed or overfixed, the increased level of crosslinking hinders the permeabilization of the tissue, resulting in an inability of the labeling reagents to penetrate the tissue,” says Uveges. “If the tissue is underfixed or fixed in the wrong fixative, the morphology of the tissue will be compromised and the location of any positive staining will not be able to be properly assessed.”
Trevigen’s TACS kits (its TUNEL assay kits) include two different permeabilization reagents. “One is the commonly used Proteinase K, a very strong permeabilization reagent that can actually damage the tissue morphology if used incorrectly,” says Uveges. “The second reagent is a milder permeabilization reagent (either Cytonin™ or Neuropore™) ideal for use in softer tissues to help maintain tissue morphology. Tissues can be kept up to a week in Cytonin without any adverse effects on tissue morphology.”
Consider detection method: Colorimetric vs. fluorescent detection
TUNEL assays generally use either colorimetric or fluorometric detection methods. Each has distinct advantages and disadvantages, so which to use depends on your particular needs. According to Uveges, fluorescent detection is more sensitive than colorimetric detection, but it is also harder to preserve the signals. “Fluorescent signals can be lost due to photobleaching during imaging and with time,” he says. “Depending on the tissue type, a fluorescent signal can also have higher background issues,” especially in mucosal-lined tissue, due to non-specific binding of the fluorescent molecule. In this situation, colorimetric detection might be preferable. “Colorimetric assays are useful for tissue sections, because they allow for use with a light microscope, and one can perform further morphological analysis that results in a permanent record,” says Almond. “Because of the sensitivity requirements, the fluorescent-based TUNEL assays are recommended for flow cytometry or fluorescent-based microplate assays.”
There are many colorimetric detection kits available, particularly for in situ or tissue research. Roche Diagnostics’ In Situ Cell Death Detection Kits use peroxidase- or alkaline phosphatase-based detection with light microscopy. Trevigen/R&D Systems offers kits specialized for different tissues that include a colorimetric detection reagent and a counterstain to give higher contrast. “For the TACS XL® kit, two colorimetric substrates are offered, TACS-Blue Label™ or DAB,” says Uveges. “TACS-Blue Label is a ready-to-use detection reagent with greater sensitivity than DAB; however, it is not a permanent stain. If the tissue and results need to be archived, DAB should be used.”
Trevigen also makes cell quantification kits based on either colorimetric or fluorescent detection. “One of the challenges with traditional TUNEL assays is the quantification of the number of apoptotic cells present in a given cell population,” says Uveges. “Counting individual cells can be tedious and time consuming. To address this need, Trevigen offers two quantifiable TUNEL assay kits, TiterTACS™ and FlowTACS™. TiterTACS™ contains the necessary reagents to generate positive controls and quantify the apoptotic signal in a given cell population, using an HRP colorimetric substrate to measure the absorbance change with a microplate reader.” TiterTACS™ is recommended for adherent cells in culture. (See below for more on FlowTACS.)
An advantage of fluorescent vs. colorimetric detection is the ability to multiplex fluorescent reactions, or run them simultaneously in the same reaction vessel. Multiplexing saves time and materials and allows for reactions to experience identical experimental conditions. This can be particularly important when trying to verify apoptosis results. “Our Click-iT® TUNEL Alexa Fluor Imaging Assays offer three different fluorescent dyes—Alexa Fluor® 488 (green), Alexa Fluor® 597 (orange), and Alexa Fluor® 647 (red) —that allow the ability to multiplex,” says Almond. “To confirm apoptosis, more than one assay is required. Thus, the ability to multiplex is essential.”
Roche offers two versions of its In Situ Cell Death Detection Kits for detection by fluorescent microscopy, using either TMR red or fluorescein. Within weeks, Roche will launch a new TUNEL-based DNA Fragmentation Imaging Kit. “It will be a simple, rapid way to measure apoptotic cell death of mammalian cells at the single-cell level using fluorescence,” says Lauren Buck, associate marketing manager for life sciences at Roche Diagnostics. “The kit is designed for use with both manual microscopy and automated imagers, such as our Cellavista Imaging system. Using this kit, total cell number in addition to the number and percentage of apoptotic cells can be determined in parallel.” Roche’s Apoptosis, Cytotoxicity, and Cell Proliferation website has an interactive product selection guide to help you determine which kit would best suit your needs.
Trevigen/R&D Systems also offers a fluorescence-based quantification tool. “FlowTACS contains the reagents to generate positive controls and detect apoptotic cells fluorescently,” says Uveges. “This kit contains a fluorescein-labeled streptavidin, and when used in conjunction with propidium iodide (as a counterstain), the number of apoptotic cells in a cell population can be quantified with a flow cytometer.” FlowTACS can be used with cells in suspension, as well as adherent cells that have been detached.
The wide range of well-optimized TUNEL assay kits available today provides researchers with a wealth of choices to find the best tools for their own experiments.
The image above is from R&D Systems' TUNEL Assays.