TUNEL Assay Kit - BrdU-Red (ab66110)

We removed flow cytometry as an application for ab66110 In situ BrdU-Red DNA Fragmentation (TUNEL) Assay Ki several years ago. We had received feedback that there was a lot of background staining and it also failed a retest in our laboratories. I think careful technique is required in order to make this work in flow cytometry.


I suggest to consider the alternative ab66108 In situ Direct DNA Fragmentation (TUNEL)Assay Kit. This has a flow cytometry procedure included in the protocol booklet:

https://www.abcam.com/ps/products/66/ab66108/documents/ab66108%20In%20situ%20Direct%20DNA%20Fragmentation%20Assay%20Kit%20v6%20(website).pdf

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.

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.

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.