Propidium iodide staining correlates with the extent of DNA degradation in isolated nuclei.
Gradual degradation of internucleosomal DNA is a hallmark of apoptosis and can be simulated by incubating isolated thymocyte nuclei in the presence of 5 mM Mg2+ and 5 mM Ca2+ at 37 degrees C. Staining of nuclei with the DNA binding fluorescent dye propidium iodide (PI) showed that intensity of fluorescence correlated with the extent of DNA degradation. PI fluorescence was increased in the presence of DNase I. Thus it seems that the cleavage of chromatin DNA by DNase 1 or by the endogenous enzyme increases the accessibility of DNA for the dye. No increase of fluorescence was observed in the presence of the known inhibitors of the endogenous endonuclease: Zn2+ and EGTA. However, the presence of Zn2+ led to decreased staining of the nuclei by PI and caused a shift in the scatter profile of the nuclei, suggesting that a conformational change of chromatin is induced by this ion. This correlation between intensity of PI staining and DNA degradation should be useful to compare endogenous nuclease levels in lymphocyte populations.
Ludwig Institute for Cancer Research, Epalinges, Switzerland.
T
T
Crompton
M C
MC
Peitsch
H R
HR
MacDonald
J
J
Tschopp
D000818:Animals; D002412:Cations / Q000494:pharmacology; D002467:Cell Nucleus / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D004249:DNA Damage; D005434:Flow Cytometry / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D011419:Propidium; D013194:Staining and Labeling; D013950:Thymus Gland / Q000166:cytology
D002412:Cations; D011419:Propidium; D004247:DNA