Apoptosis is a genetically programmed process that ensures proper elimination of unwanted or diseased cells. As such, it is essential for normal development and homeostasis. Deregulation of apoptosis is a hallmark of various pathologies including cancer, Alzheimer’s disease and AIDS. Unlike the passive process of necrotic cell death, apoptosis is an active process requiring the activation of various proteases and nucleases which ultimately lead to cell suicide. For instance, nuclease activation by apoptotic stimuli, such as serum deprivation of LPS, cleaves naked double-stranded DNA resulting in mono- and oligo-nucleosomal fragments which cannot be further processed due to the DNA/histone complex of nucleosomes. These fragments were traditionally detected by gel electrophoresis of DNA samples, which is a qualitative but non-quantitative method that requires large amounts of sample and an “all or none” stimuli.
Roche’s Cell Death Detection ELISAPLUS kit qualitatively and quantitatively detects the amount of cleaved DNA/histone complexes (nucleosomes) in a given sample using a sandwich-enzyme-immunoassay-based method. It is very straightforward to use with any type of sample, from cell lysates to serum to whole tissues. Samples are placed on a streptavidin-coated 96-well plate and then incubated with monoclonal antibodies against both histone-biotin and DNA-POD. The anti-histone-biotin and anti-DNA-POD bind to the histone and DNA component of nucleosomes in the samples, respectively, with the biotin moiety “capturing” nucleosomes onto the plate. After washing away unbound samples and reagents, the amount of captured histone is detected colorimetrically following the reaction between the POD moiety and the ABTS substrate.
Although generally a well-optimized kit, there are a few disadvantages, mainly the need to first optimize the amount of sample one should use for each type of sample (e.g. for each cell type and condition). A background control should also be prepared since the background of this kit is inherently high. We did not always obtain background values which were less than 100mU, as indicated in the brochure. Although the company claims that the ABTS substrate is stable for 1 month once dissolved, I recommend always using a fresh preparation of the substrate in order to further prevent high background.
A main advantage of this kit is the low amount of sample required, provided you have adequate apoptotic induction. For example, 5g of tissue from LPS-treated rats or 40g of serum deprived cell lysates were sufficient to detect a significant signal as opposed to their respective controls. The kit also gave highly reproducible results for both cells and tissues. In addition, very few pieces of specialized equipment are needed. Although a spectrophotometer is required, the readings do not have to be done at the recommended 405 nm; readings at 450 – 660nm (in older spectrophotometers) are adequate. A regular orbital shaker (medium speed) can be used instead of the recommended MP shaker.
In conclusion, this kit is a must for any lab routinely performing apopotosis assays and wanting quantitative as well as qualitative measurements. This kit greatly complements other standard apoptotic assays such as caspase enzymatic assays and immunoblotting and can easily replace the more traditional DNA ladder gels.
Rania Harfouche
PhD Candidate
McGill University Health Center