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TUNEL Assays

Internucleosomal DNA fragmentation caused by activated endonucleases is universally regarded as the biochemical hallmark of apoptosis. Cells containing these DNA strand breaks can be identified with the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. This well-established in situ staining method relies on the enzyme terminal deoxynucleotidyl transferase (TdT) to catalyze the incorporation of modified dUTPs to the 3'-hydroxyl termini of fragmented DNA. Depending upon the choice of modified dUTP - BrdUTP, biotin-dUTP, or a fluorescein-dUTP - apoptotic cells can be identified and measured using various detection strategies and systems, including fluorescence microscopy, flow cytometry, and fluorescence-based microplate studies.

AAT Bioquest offers several fluorimetric TUNEL assays optimized for in situ apoptosis detection in live cells or fixed cells and tissue samples. These products can be combined with other cell-based viability and apoptosis assays to provide a comprehensive assessment of cell health, assess toxicity and safety of drug candidates, or analyze cancer and disease-related cellular changes.

 



TUNEL Assay Principles


During the later stages of apoptosis, caspase-activated endonucleases cleave genomic DNA into oligonucleosomal fragments (∼ 180-200 base pairs long). Using the TUNEL assay, the exposed 3'-OH termini of these breaks can be marked with modified dUTPs for subsequent visualization and quantification of apoptotic cells in situ. This is generally done either directly using dye-modified dUTP or indirectly using BrdUTP and antibody conjugates against BrdUTP. Regardless of the labeling strategy, both require the enzyme terminal deoxynucleotidyl transferase (TdT) to catalyze the incorporation of the modified dUTPs to the 3'-OH termini.

Detection of DNA fragments by this assay requires sample fixation using a crosslinking reagent such as 4% paraformaldehyde (avoid ethanol-based fixatives as it hinders the extraction of small DNA fragments) and sample permeabilization. Both steps are critical to the TUNEL assay as it facilitates the entry of exogenous TdT and antibody conjugates against BrdUTP. Keep in mind that several variables influence the staining kinetics of the TUNEL assay. Some of these variables, including reagent concentration, fixation of the sample, and accessibility of DNA strand breaks, may vary between tissue or cell sample types. Standardizing the TUNEL assay using samples with positive and negative apoptosis controls can alleviate these potential influences and reduce false positives or negative results.

 

Figure 1. Detection of apoptosis with the Cell Meter™ Fixed Cells and Tissue TUNEL Apoptosis imaging assay.

 



TUNEL Assays for Live Cells


Traditionally, TUNELs assays require fixation and permeabilization of the sample to facilitate the entry of exogenous TdT and ensure successful labeling of DNA strand breaks. While critical to the outcome, this additional handling requires added hands on-time and optimization of conditions, so results are accurate and reproducible. The Cell Meter™ Live Cell TUNEL Apoptosis Assays are designed to identify DNA fragmentation without the enzyme TdT and the additional handling that comes with its use. These assays utilize a proprietary membrane-permeant fluorescent dye that passively enters live cells and selectively targets the nicks in DNA that form during apoptosis. The fluorescently-labeled DNA fragments can then be visualized directly by fluorescence microscopy, flow cytometry, or fluorescence-based microplate assays. The Cell Meter™ Live Cell TUNEL Apoptosis assays are available in two emission colors (green and red) for flexibility in multiparametric analysis with other fluorescence-based cell function assays, such as caspase activity assays and annexin V staining assays.

 

Control
Control
SS 100 nM
SS 100 nM
SS 1 µM
SS 1 µM
Figure 2. Fluorescence images of TUNEL reaction in HeLa cells with the treatment of 100 nM or 1 µM staurosporine (SS) for 4 hours as compared to untreated control. Cells were incubated with a TUNEL working solution for 1 hour at 37°C. The red fluorescence signal was analyzed using a fluorescence microscope with a TRITC filter set. Fluorescently labeled DNA strand breaks show intense fluorescent staining in SS-treated cells.

 

Table 1. Cell Meter™ TUNEL assays for live cell analysis

Assay
Ex/Em (nm)¹
Cutoff (nm)¹
Filter Set²
Channel³
Unit Size
Cat No.
Cell Meter™ Live Cell TUNEL Apoptosis Assay Kit *Green Fluorescence*490/525515FITC Filter SetFITC Channel25 tests22849
Cell Meter™ Live Cell TUNEL Apoptosis Assay Kit *Red Fluorescence*550/590-650570TRITC Filter SetPE-Cy5 Channel25 tests22844
  1. Fluorescence microplate instrument specifications.
  2. Fluorescence microscope instrument specifications.
  3. Flow cytometer instrument specifications.



TUNEL Assays for Fixed Cells and Tissue Samples


The Cell Meter™ Fixed Cell and Tissue TUNEL Assays follow the traditional TUNEL method and are optimized for in situ apoptosis detection in both fixed cells and tissue sections. These assays directly identify apoptotic cells within a population by using TdT to catalyze the incorporation of dye-modified dUTPs at the 3'-OH termini of fragmented DNA. The fluorescently-labeled DNA fragments can then be visualized or quantified using fluorescence microscopy or flow cytometry, respectively. The Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis assays are available in four emission colors (blue, green, red, and deep red) for flexibility in multiparametric analysis with other fluorescent proteins or conjugates, such as phalloidin.

Key Features

  • Validated for apoptosis detection in fixed cells and formalin-fixed, paraffin-embedded tissue sections
  • Direct incorporation minimizes hands on-time by reducing the number of incubation steps
  • Compatible with other fluorescent proteins and conjugates for multiplex analysis
  • The assay provides sufficient reagents for 25 tests

 

Control
No DNase
SS 100 nM
DNase
Figure 2. TUNEL assay of formalin-fixed paraffin-embedded (FFPE) human lung adenocarcinoma sections with Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit *Green Fluorescence*. DNA strand breaks showed intense fluorescent staining in DNAse treated tissue section. The cell nucleus was stained with Nuclear Blue™ DCS1 (Cat No. 17548).

 

Table 2. Cell Meter™ TUNEL assays for fixed cells and tissue analysis

Assay
dUTP
Ex/Em (nm)¹
Channel¹
Filter Set²
Unit Size
Cat No.
Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit *Blue Fluorescence*DEAC-dUTP405/525Pacific Orange ChannelViolet Filter Set25 tests22857
Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit *Green Fluorescence*Fluorescein-12-dUTP488/530FITC ChannelFITC Filter Set25 tests22851
Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit *Red Fluorescence*TMR-dUTP488/575PE ChannelCy3/TRITC Filter Set25 tests22853
Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit *Deep Red Fluorescence*TF5-dUTP633 or 640/660APC ChannelCy5 Filter Set25 tests22855
  1. Flow cytometer instrument specifications.
  2. Fluorescence microscope instrument specifications.



Product Ordering Information



Table 3. TUNEL Apoptosis assays for live cells, fixed cells or tissues.

Product
Unit Size
Cat No.
Cell Meter™ Live Cell TUNEL Apoptosis Assay Kit *Green Fluorescence*50 Tests22849
Cell Meter™ Live Cell TUNEL Apoptosis Assay Kit *Red Fluorescence*50 Tests22844
Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit *Green Fluorescence*25 Tests22851
Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit *Red Fluorescence*25 Tests22853
Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit *Deep Red Fluorescence*25 Tests22855
Cell Meter™ Fixed Cell and Tissue TUNEL Apoptosis Assay Kit *Blue Fluorescence*25 Tests22857