Is it possible to differentiate between apoptotic and necrotic cells using the CometAssay®?
FAQ from Trevigen
Listed below are two references discussing differentiation of apoptotic and necrotic cells using a derivation of the comet assay.
Fairbairn, D. W., Walburger D. K, Fairbairn J. J and O'Neill K. L. Key morphologic changes and DNA strand breaks in human lymphoid cells: discriminating apoptosis from necrosis. Scanning 1996 Sep: 18(6):407-16
Apoptosis is an important form of physiologic cell death displayed by an enormous variety of tissues under divergent conditions. The recent attention toward apoptosis in virtually all aspects of modern biology indicates that rapid and accurate differentiation between apoptosis and necrotic death is of considerable interest.
Apoptosis is distinguishable from necrosis on the basis of several criteria.
In this study, we undertook to examine the effects of mild hyperthermia (43 degrees C leading to apoptotic death) and severe hyperthermia (50 degrees C leading to necrotic killing) on associated DNA fragmentation. Using laser scanning and fluorescent microscopic evaluation of DNA "comets" in the single cell gel assay, we compared necrotic and apoptotic DNA damage in a variety of human leukemia and lymphoma cell lines at the level of the individual cell.
We show that necrotic cells do display detectable DNA damage. We confirm our preliminary report that comet "tail moment" is sufficient to distinguish between necrotic and apoptotic DNA damage, while comet tail length may confuse the two forms. We report that recovery period is necessary for expression of increasing apoptotic but not necrotic DNA damage.
We show that apoptosis increases with prolonged hyperthermia and confirm that the mode of death changes from apoptosis to necrosis with higher heat loads, producing a greater fraction of cells showing damage.
In addition, we show that for necrotic cells, DNA tail moment reflects sensitivity to prolonged exposure without a concomitant change in tail length.
Singh, N. P., A simple method for accurate estimation of apoptotic cells. Exp Cell Res 2000 Apr 10; 256(1):328-37
A simple, sensitive, and reliable "DNA diffusion" assay for the quantification of apoptosis is described.
Human lymphocytes and human lymphoblastoid cells, MOLT-4, were exposed to 0, 12.5, 25, 50, or 100 rad of X-rays.
After 24 h of incubation, cells were mixed with agarose, microgels were made, and cells were lysed in high salt and detergents. DNA was precipitated in microgels by ethanol. Staining of DNA was done with an intense fluorescent dye, YOYO-1. Apoptotic cells show a halo of granular DNA with a hazy outer boundary.
Necrotic cells, resulting from hyperthermia treatment, on the other hand, show an unusually large homogeneous nucleus with a clearly defined boundary. The number of cells with apoptotic and necrotic appearance can be scored and quantified by using a fluorescent microscope. Results were compared with other methods of apoptosis measurement: morphological estimations of apoptosis and DNA ladder pattern formation in regular agarose gel electrophoresis.
Validation of the technique was done using some known inducers of apoptosis and necrosis