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Investigación en Ciencias de la Vida
 

DNA Damage & TUNEL Assays

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Illustration of DNA damage.
DNA fragmentation in apoptosis is usually associated with ultra-structural changes in cellular morphology. In a number of well-researched model systems, large fragments of 300 kb and 50 kb are first produced by endonucleolytic degradation of higher-order chromatin structural organization. DNA fragmentation in apoptosis can be examined using the TUNEL assay. The in situ staining of DNA strand breaks detected by the TUNEL assay and subsequent visualization by light microscopy gives biologically significant data about DNA damage, as it is related to the apoptotic process.

DNA fragmentation detection via the TUNEL assay is the basis of our ApopTag® technology. The DNA strand breaks are detected by enzymatically labeling the free 3'-OH termini with modified nucleotides. These new DNA ends that are generated upon DNA fragmentation are typically localized in morphologically identifiable nuclei and apoptotic bodies. In contrast, normal or proliferative nuclei, which have relatively insignificant numbers of DNA 3'-OH ends, usually do not stain with the kit.

ApopTag Kits detect single-stranded and double-stranded breaks associated with apoptosis. Drug-induced DNA damage is not identified by the TUNEL assay unless it is coupled to the apoptotic response. In addition, this technique can detect early-stage apoptosis in systems where chromatin condensation has begun and strand breaks are fewer, even before the nucleus undergoes major morphological changes.

Merck is the leader in providing well-published, all-in-one, and optimized TUNEL assays that bring consistency and convenience to apoptosis research.

Product Highlight:
ApopTag Fluorescein In Situ Apoptosis Detection Kit
ApopTag Fluorescein In Situ Apoptosis Detection Kit
Detection of apoptotic cells with ApopTag Fluorescein Direct kit in mouse embryo forelimb bud, fixed with 10% neutral-buffered formalin and paraffin-embedded.
The ApopTag Fluorescein In Situ Apoptosis Detection Kit detects apoptotic cells in situ by the indirect TUNEL method, utilizing an anti-digoxigenin antibody that is conjugated to a Fluorescein reporter molecule. It provides indirect immunofluorescence staining and results can be analyzed using either flow cytometry or fluorescence microscopy.

The ApopTag Fluorescein In SituApoptosis Detection Kit has been tested for specific staining in these model systems: (a) human normal peripheral blood lymphocytes induced with dexamethasone as stained in cytospins, (b) rat regressing mammary gland as stained in formalin-fixed, paraffin-embedded sections, and (c) human leukemic peripheral blood lymphocytes induced with camptothecin, as stained in cell suspensions and used for quantitative flow cytometry.

Product Highlight:
ApopTag ISOL Dual Fluorescence Apoptosis Detection Kit (DNase Types I & II)
Detection of apoptotic and phagocytic cells in paraffin-embedded rat mammary gland tissue.
Detection of apoptotic and phagocytic cells in paraffin-embedded rat mammary gland tissue sections using ApopTag ISOL Dual Fluorescence Apoptosis Detection Kit (DNase Types I & II).
While conventional in situ detection techniques such as ISEL (Klenow DNA polymerase), TUNEL (terminal deoxynucleotidyl transferase, TdT) and ISNT (DNA Polymerase I) are useful in detecting internucleosomal DNA cleavage, they do not differentiate DNase Type I and DNase Type II cleavage which results from the activation of apoptotic endonucleases. ApopTag ISOL Kits facilitate the differentiation of apoptotic cells from necrotic or transiently damaged cells.

The ApopTag ISOL Dual Fluorescence Kit utilizes a proprietary double hairpin, dual fluorescently labeled oligonucleotide labeling process to detect and distinguish between typical apoptotic DNA breaks induced by either DNase I or DNase II enzyme activities. The Vaccinia Topoisomerase I mediated ligation reaction is adaptable for staining paraffin-embedded tissue, frozen tissue sections, cell suspensions, and adherent cells. In situ staining for DNA fragmentation (as in the ISOL method) is both a means of detection for rare cells and an analytical test of those cells' DNA,

Product Highlight:
Apo-Direct TUNEL Assay Kit
Apo-Direct TUNEL Assay Kit
Detection of apoptotic cells via flow cytometry using the Apo-Direct TUNEL Assay Kit. (Click to enlarge)
The Apo-Direct Kit is a single-step staining method for labeling DNA breaks to detect apoptotic cells by flow cytometry. The kit contains all the reagents required for measuring apoptosis in cells including positive and negative control cells; washing, reaction, and rinsing buffers; and propidium iodide/RNase A solution for counter staining the total DNA.


Merck's Family of TUNEL Assays

DescriptionCatalog No.Assays
ApopTag Red In Situ Apoptosis Detection Kit S7165 40 assays
ApopTag Plus Peroxidase In Situ Apoptosis Kit S7101 40 assays
ApopTag Fluorescein Direct In Situ Apoptosis Detection Kit S7160 40 assays
ApopTag Peroxidase In Situ Apoptosis Detection Kit S7100 40 assays
ApopTag Fluorescein In Situ Apoptosis Detection Kit S7110 40 assays
ApopTag Plus In Situ Apoptosis Fluorescein Detection Kit S7111 40 assays
Apo-Direct TUNEL Assay Kit APT110 50 assays
ApopTag ISOL Dual Fluorescence Apoptosis Detection Kit (DNase Types I & II) APT1000 25 assays
FragEL DNA Fragmentation Detection Kit, Colorimetric - Klenow Enzyme QIA21 50 assays
FragEL DNA Fragmentation Detection Kit, Colorimetric - TdT Enzyme QIA33 50 assays
FragEL DNA Fragmentation Detection Kit, Fluorescent - TdT Enzyme QIA39 50 assays

Whether cells are in the early stages of apoptosis (with exposed inner leaflets), intermediate stages (with active caspase activity), or late stages (with extensive DNA fragmentation), Merck’s reagents and cell-based assays detect specific cell death processes across the apoptotic timeline.