Dysfunction in the apoptotic pathway is a hallmark of many diseases such as cardiovascular disease, cancer, autoimmune disorders and neurodegenerative diseases. Additionally, apoptosis may be induced in response to various “death signals” including activation of cell surface receptors by TNF-alpha and Fas, UV irradiation or treatment with DNA damaging agents. The apoptotic program is characterized by membrane blebbing, condensation of cytoplasm and the activation of endogenous endonucleoses. This leads to internucleosomal cleavage of DNA and the generation of mono- and oligonucleosomes, which are tightly complexed with histones. The DNA contained in these nucleosomes is protected from cleavage since it is tightly bound to the histones.
The Roche Cell Death Detection ELISA kit is based on the quantitative detection of histone-associated DNA fragments in mono- and oligonucleosomes (a marker for apoptotic cells). The biggest advantage of the kit is that it is designed for a 96-well plate format, hence multiple samples can be run in a single assay. The Cell Death Detection ELISA kit is highly sensitive and detects apoptotic nucleosomes in cell lysates and tissue homogenates, which requires fewer cells than flow cytometry-based apoptosis assays. This aspect is especially useful when one is doing experiments to study apoptosis in slow-growing primary cells and patient tissue samples, which are hard to obtain in large quantities. The kit works well for both adherent and suspension cells as well as a variety of cell lines including mouse, rat and xenopus. The assay is simple to perform and one can obtain results within 5-6 hours. The instruction manual suggests positive and negative controls that may be used in the assay; furthermore, it gives detailed instructions on how to perform the assay as well as calculate and interpret the results.
The Cell Death Detection ELISA kit is highly sensitive, thus experiments should be done using healthy cells as the negative control. If the cells are stressed during routine culture conditions, the negative control itself can give substantial background. This should not pose a problem except when apoptosis experiments are performed with quiescent cells restimulated with growth factors (the background can be significant). Therefore, it is essential to optimize experimental conditions so that the background absorbance is low. The numerous advantages of the Roche Cell Death Detection ELISA Kit, make it a valuable asset for research laboratories in the field of apoptosis research.
Piyali Dasgupta, Ph.D.
Post-Doctoral Fellow
Moffit Research Center