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Your are here: Products & Ordering / Nucleosides, Nucleotides and their Analogs / Nucleotides / Nucleotides by Application / ...in Cell Biology / Apoptosis
(TUNEL assay)

...in Cell Biology - Apoptosis (TUNEL assay)
Apoptosis is the process of an intracellular death program leading to characteristic biochemical and morphological changes within a
cell that consequently result in cell death
[1]
. A failure of cells to undergo apoptosis is a common feature of many cancers [2] thus
investigation of apoptosis inducing mechanisms is of particular importance for cancer research and requires suitable detection
methods.
A hallmark of late apoptosis is extensive genomic DNA fragmentation that generates a multitude of DNA double-strand breaks
(DSBs) with accessible 3'-hydroxyl (3'-OH) groups. This characteristic forms the basis for a well-established apoptosis detection
method: Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) assay
[3]
.
TUNEL assays identify apoptotic cells by the terminal deoxynucleotidyl transferase (TdT)-mediated addition of labeled (X)
deoxyuridine triphosphate nucleotides (X-dUTPs) to the 3-OH end of DNA strand breaks that are subsequently visualized
depending on the introduced label (Fig. 1), thus serving as parameter for the percentage of apoptotic cells within the analyzed cell
population.
The assay sensitivity strongly depends on the incorporation efficiency of the modified dUTP that is influenced by size/bulkiness of
the attached label. A number of fluorescently labeled dUTPs, biotinylated dUTPs or digoxigenylated dUTPs are generally suitable
substrates for TdT, but dUTP labeled with smaller labels such as bromine (BrdUTP) or an alkyne group (EdUTP) have been
demonstrated to exhibit a higher incorporation efficiency and thus higher sensitivity in TUNEL assays probably due to minimal
sterical hindrance
[3,4,5]
.
While incorporation of BrdUTP is detected by specific fluorophore or reporter enzyme labeled antibodies (Fig. 1A), detection of
EdUTP is achieved by covalent conjugation of an azide-containing fluorophore via Cu(I) catalyzed alkyne-azide click chemistry
(CuAAC) (Fig. 1B).
Figure 1: The principle of TUNEL assay relies on terminal deoxynucleotidyl transferase (TdT)-mediated addition of a modified dUTP (X-dUTP)
to 3-OH ends of DNA fragments that are generated as a result of apoptosis induction. To avoid the loss of fragmented DNA and to allow enzyme
and nucleotide entrance, cells need to be fixed and subsequently permeabilized prior to the labeling reaction. Incorporated bromoylated dUTP
(BrdUTP) is detected by specific antibody conjugates with a reporter enzyme or fluorescent dye (A). The incorporation of alkyne-containing
dUTP (EdUTP) is visualized by Cu(I)-catalyzed alkyne-azide click chemistry (CuAAC) with an azide containing fluorphore (B).
5Br-dUTP
Product Cat. No. Amount Price (EUR) Buy / Note
S pack NU-122S 50 Units 115,56

L pack NU-122L 5x 50 Units 338,49

5-Ethynyl-dUTP (5-EdUTP)
Product Cat. No. Amount Price (EUR) Buy / Note




Apoptosis (TUNEL assay), ...in Cell Biology, Nucleotides by Applicatio... http://www.jenabioscience.com/cms/en/1/catalog/2205_apoptosis_tunel_...
1 of 2 5/13/2014 6:16 PM
S pack CLK-T07-S 0,5 mol 122,69

M pack CLK-T07-M 2,5 mol 490,77

L pack CLK-T07-L 5 mol 736,16

Selected References
[1] Kerr et al. (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer
26(4):239.
[2] Lowe et al. (2000) Apoptosis in Cancer. Carcinogenesis 21(3):485.
[3] Darzynkiewicz et al. (2008) Analysis of apoptosis by cytometry using TUNEL assay. Methods 44(3):250.
[4] Li et al. (1995) Labelling DNA strand breaks with BrdUTP: Detection of apoptosis and cell proliferation. Cell proliferation
28(11):571.
[5] Li et al. (1995) Singlestep procedure for labeling DNA strand breaks with fluorescein- or BODIPY-conjugated deoxynucleotides:
Detection of apoptosis and bromdeoxyuridine incorporation. Cytometry 20:172.
1 Unit = 1 l of a 10 mM solution.
Please contact nucleotides@jenabioscience.com with questions or inquiries.


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Apoptosis (TUNEL assay), ...in Cell Biology, Nucleotides by Applicatio... http://www.jenabioscience.com/cms/en/1/catalog/2205_apoptosis_tunel_...
2 of 2 5/13/2014 6:16 PM