A novel method for quantitative analysis of apoptosis.
In the present study, we describe a method of quantifying DNA fragmentation. This assay is based on saturation labeling 3'-ends of DNA fragments with alpha(32)PdCTP in the presence of 2',3'-dideoxy-cytidine-5'-triphosphate (ddCTP) by terminal deoxynucleotidyl transferase (TdT). The saturation labeling of 3'-ends of DNA fragments was performed by adding different concentrations of alpha(32)PdCTP to a DNA sample, from which a maximal labeling (Lmax) and a kinetic parameter (Km) of the TdT reaction are calculated. The saturated labeling gives true quantitation that makes it possible to accurately compare quantities of DNA fragments among different samples. This method requires as little as 5 ng of DNA and increases the sensitivity of apoptotic DNA detection by at least 200-fold relative to the widely used ethidium staining method. The application of this method in an apoptosis study showed that (a) a time- and dose-dependent increase in the number of DNA strand breaks in apoptotic lymphocytes was induced by dexamethasone, and (b) age-dependent apoptosis occurred in the cardiac tissues of spontaneously hypertensive rats. Results of this assay were confirmed by the DNA ladder pattern exhibited after electrophoresis as well as the morphologic changes of apoptosis observed under electron microscopy and were very consistent with results obtained in quantifying apoptotic cells by flow cytometric analysis (r = 0.98, p = 0.002). Thus, this assay is quantitative, simple, sensitive, and useful for assessing apoptosis.
Peng
L
L
Vascular Biology Unit, University of Melbourne, Heidelberg, Victoria, Australia.
Liu
J J
JJ
D000818:Animals; D017209:Apoptosis; D004249:DNA Damage; D006801:Humans; D008722:Methods; D051381:Rats