Overview

  • Product name
    TUNEL Assay Kit - BrdU-Red
    See all DNA fragmentation kits
  • Detection method
    Fluorescent
  • Sample type
    Tissue, Adherent cells, Suspension cells
  • Assay type
    Quantitative
  • Assay time
    3h 00m
  • Species reactivity
    Reacts with: Other species, Mammals
  • Product overview

    TUNEL Assay Kit - BrdU-Red ab66110 (previously called TUNEL Assay Kit - In situ BrdU-Red DNA Fragmentation) uses a convenient and sensitive method to detect DNA fragmentation by flow cytometry and fluorescence microscopy in live cells.


    This TUNEL assay protocol uses Br-dUTP (bromolated deoxyuridine triphosphate nucleotide), which can be more readily incorporated into DNA strand breaks by the TdT enzyme than other dUTP labels such as fluorescein, biotin or dioxigenin. The greater incorporation rate produces a brighter signal when the Br-dUTP sites are detected with an anti-BrdU monoclonal antibody directly labeled with a red fluorochrome.


    BrdU-Red can be analyzed at Ex/Em 488/576 nm, with an optional 7-AAD counterstain at Ex/Em 488/655nm.


    The TUNEL assay kit includes both negative and postive control cells. It is designed to be suitable for studying DNA fragmentation in GFP-transfected cells.


    Tunel assay protocol summary:
    - fix cells / tissues with formaldehyde, or deparaffinize and rehydrate if paraffin sections, and wash
    - incubate cells in 70% ethanol for 30 min at 4ºC, or if tissues incubate with proteinase K solution for 5 min at room temp and refix with formaldehyde
    - wash
    - incubate in DNA labeling solution for 60 min at 37ºC
    - wash
    - incubate in antibody solution for 30 min at room temp
    - add 7-AAD / RNase A solution and incubate for 30 min at room temp
    - analyze with flow cytometry or fluorescent microscopy

  • Notes

    This kit is BrdU-Red labeled (Ex/Em = 488/576 nm).

    To use FITC (Ex/Em = 495/519 nm) as a label, we recommend TUNEL Assay Kit - FITC (ab66108).

    For chromogenic TUNEL staining, we recommend TUNEL Assay Kit - HRP-DAB ab206386.

    Find out more about the TUNEL method in the TUNEL staining / TUNEL assay guide.

  • Platform
    Flow cytometer, Fluorescence microscope

Properties

Images

  • Immunohistochemical analysis of paraffin embedded 5 micron thick testis tissues of 8 week old Stag3+/− and Stag3−/− (Stromal antigen) mice . Apoptotic cells were detected using In situ BrdU-Red DNA Fragmentation (TUNEL) Assay Kit (ab66110). DAPI was used as a counterstain.

  • Detection of DNA fragmention (TUNEL staining) using the negative and positive control cells (HL-60 untreated and treated with camptothecin). Cells were stained following the assay protocol. The fluorescence signal was detected and analyzed using BD FACScan System (Becton Dickinson).

  • TUNEL staining in whole mount Hydractinia echinata using In situ BrdU-Red DNA Fragmentation (TUNEL) Assay Kit (ab66110).

    Animals were fixed in 4% PFA in PBS for 1 hour and processed as per the protocol without proteinaseK treatment. In place of proteinaseK animals were permeabilised in 3% Triton in PBS for 15 minutes. Animals were counter-stained with DAPI.

Protocols

References

This product has been referenced in:
  • Zhang B  et al. Mesenchymal stem cells rejuvenate cardiac muscle after ischemic injury. Aging (Albany NY) 11:63-72 (2019). Read more (PubMed: 30613028) »
  • Chen YJ  et al. Inhibition of the potassium channel Kv1.3 reduces infarction and inflammation in ischemic stroke. Ann Clin Transl Neurol 5:147-161 (2018). Read more (PubMed: 29468176) »
See all 17 Publications for this product

Customer reviews and Q&As

1-10 of 14 Abreviews or Q&A

TUNEL staining on Lewis Lung Carcinoma cells in vitro

Excellent Excellent 5/5 (Ease of Use)
Abreviews
Worked very well on mouse Lewis Lung Carcinoma cells grown in vitro.

Dr. Louise Reynolds

Verified customer

Submitted Apr 05 2019

Excellent on human tumor tissue

Excellent Excellent 5/5 (Ease of Use)
Abreviews
This kit is very good when I used on human tumor sections. Just follow the manual and you will get good results. Also, if you want do double fluorescent staining for other antigen, you can use heat induced antigen retrieve too.

Dr. Hongwei Shao

Verified customer

Submitted Dec 19 2018

Abreviews
It works on quail tissue.
It is a long procedure, it fades relatively fast.

Abcam user community

Verified customer

Submitted Nov 28 2018

Flow Cyt

Average Average 3/5 (Ease of Use)
Abreviews
I tried to used (ab66110) For the Flow Cyt after fixing the cells it dose not give any differences between the positive and negative samples (dose not work)

Abcam user community

Verified customer

Submitted Nov 15 2018

Abreviews
I purchased this kit with the purpose of staining cultured cells. It totally failed in that purpose and I would recommend the xxxx kit if your aim is to investigate apoptosis in cultured cells.
The protocol provided by Abcam does not mention any suggestion for cultured cells.
Additionally, the protocol is not well written. This includes a mistake on the manual with a recommended amount to pipette. You will easily find the mistake but this should not happen. I sent an email to Abcam and I hope they correct the mistake.

On the other hand, the kit works pretty well for tissues embedded in Paraffin. Attached is an example of mouse mammary gland. Magnification = 20X

Abcam user community

Verified customer

Submitted Sep 08 2017

Dual Caspase-3/TUNEL Fluorescence Staining

Excellent Excellent 5/5 (Ease of Use)
Abreviews
Frozen sections of snap frozen sekeletal muscle tissue with isopentane were used in the application. Sections were fixed in cold acetone for 10 minutes and then treated with 5% normal goat serum as a blocking reagent for 30 minutes.

*PBS was used for rinsing/washing solution in between steps.

No antigen retrieval used.

PART A: Caspase-3
i. Primary antibody ab52293 anti-Caspase-3 (1/200, overnight incubation at 4C)
ii. Secondary antibody ab7086 Goat anti-Rabbit IgG H&L FITC (1/200, 1 hour incubation at RT)

PART B: TUNEL
i. As per kit protocol from Part 2 (Detection by Fluorescence Microscopy), steps (a) to (l)
ii. After step (l), sections were dipped in DAPI diluted in PBS (1/10000) for 1 minute prior to final washing steps

Sections were coverslipped using fluoromount mountant and kept refridgerated at 4C in the dark until ready for visualisation.

Abcam user community

Verified customer

Submitted Apr 07 2016

Answer

We have not done any additional antibody staining with the ab66110 In situ BrdU-Red DNA Fragmentation (TUNEL) Assay Kit .Whether it can be done will depend on the criteria required by the additional antibody but theoretically staining should work after the ApoBrdU labeling.


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Abreviews
Animals were fixed in 4% PFA in PBS from 1 hour and processed as per the protocol without proteinaseK treatment. In place of proteinaseK animals were permeabilised in 3%Triton in PBS for 15 minutes. Animals were counter-stained with DAPI.

Mr. James Gahan

Verified customer

Submitted Oct 06 2014

Answer

This kit is specifically designed for and has been used for co detection of GFP transfected cells and the Red fluorescence labeled anti-BrdU monoclonal antibody. There is therefore no need to use this other antibody conjugated to AlexaFluor647.

When doing this assay, please read at the following wavelengths:

Ex/Em = 488/576 nm for BrdU-Red (Rhodamine dye). The PE channel PMT (photomultiplying tube) will detect light emitted at 575 nm wavelength so should be used for BrdU-Red
Ex/Em = 488/507 nm for GFP


For more information regarding compensation in flow cytometry please see our following webpage: https://www.abcam.com/index.html?pageconfig=resource&rid=13433

The lab have also suggested that you can have GFP only and BrdU-Red only controls and excite with 488nm wavelength and measure emission at both 507 and 576 nm to make sure there is no significant overlap/spillover. These following citations which use a similar kit may also help:


Apo-BrdU DNA Fragmentation Assay Kit

- Cassano, M. et. al. Alpha sarcoglycan is required for FGF-dependent myogenic progenitor cell proliferation in vitro and in vivo. http://dev.biologists.org/content/138/20/4523.abstract?maxtoshow=&HITS=10&hits=50&RESULTFORMAT=1&andorexacttitle=and&andorexacttitleabs=and&fulltext=Biovision&andorexactfulltext=and&searchid=1&FIRSTINDEX=0&sortspec=match&fdate=10/1/2011&tdate=10/31/2011&resourcetype=HWCIT

- Cruz AR et al (2008) Infect. Immun. 76: 56 - 70

- Kikuchi, J.. et al.Histone Deacetylases Are Critical targets of Bortezomib-Induced Cytotoxicity in Multiple Myeloma. Blood, 2010; 116: 406-417.

- Sheu JJ-C et al (2008) Cancer Res. 68: 4050 - 4057.

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Question
Answer



The 7AAD stain has different excitation and emission maxima than the Red fluorescence from the antibody ( emission max is 576 versus 655nm). This difference is enough to distinguish spectrally the signal from the antibody and 7AAD. The main reason for selection of 7-AAD is that conventional bench-top analyzers utilize a 488-nm laser line, which can excite DNA stains such as 7-amino-actinomycin D (7-AAD) but not DAPI. The availability of an ultraviolet (UV) laser line to excite DAPI is not commonly available on the same instrument in labs. This is why 7-AAD has greater flexibility and ease of use.

Flow cytometry offers a more definitive and quantitative way to look at the cells in comparison to microscopy. As stated on page 11 of the protocol booklet, the quantification by flow cytometry protocol can be used for your test cells and the control cells. Part 2 of the protocol is the same for control cells as well. You need to carry out step 2i and then only the control cells are labelled with the antibody for visualization.

To use microscopy, simply follow this protocol until you reach part 2l and rather than analysing on a flow cytometer, cells can be aliquoted onto a glass slide, covered with a coverslip and observed under a fluorescence microscope. There is no complicated protocol steps to provide. Typically 50 ul cells should be enough to be put on the slide. The correct excitation/emission filters need to be used for visualization.

The positive control cells have been induced to initiate DNA fragmentation and hence should show higher Brd-UTP labeling.

Here are some more technical tips for ab66110 and our TUNEL assays in general:

1. For adherent cell line systems, the cells in the supernatant have a higher probability of being apoptotic than do the adherent cells. Save cells in the supernatant for assay prior to trypsinization of the adherent cell layer.

2. Cell fixation using a DNA crossing linking chemical fixative is an important step in analyzing apoptosis. Unfixed cells may lose smaller fragments of DNA that are not chemically fixed in place inside the cell during washing steps. The researcher may have to explore alternative fixation and permeablization methods to fully exploit their systems.

3. To minimize cell loss during the assay, restrict the assay to the use of a single 12 X 75 mm test tube. If polystyrene plastic test tubes are used, an electrostatic charge can build up on the sides of the tube. Cells will adhere to the side of the tube and the sequential use of multiple tubes can result in significant cell loss during the assay.

4. Occasionally a mirror image population of cells at lower intensity is observed in the flow cytometry dual parameter display. This population arises because during the 50 μl DNA Labeling Reaction some cells have become stuck to the side of the test tube and are not fully exposed to the reaction solution. This phenomenon can be overcome by washing all the cells from side of the tube and making sure all cells are properly suspended at the beginning of the labeling reaction.

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1-10 of 14 Abreviews or Q&A

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