Qualitative and quantitative analysis of DNA fragmentation using digital imaging.
Apoptosis is an important and common pathway of cellular death. Differentiation from cellular necrosis and quantitation of apoptosis within the milieu of necrosis are analytical challenges. We describe the use of the RIT120 digital imaging software package for quantitative and qualitative analysis of apoptotic DNA ladders induced by a variety of agents, such as serum, tumor necrosis factor-alpha, transforming growth factor-beta1, and nitric oxide. Autoradiographs of DNA ladders are densitometrically scanned to yield a set of curves with peaks corresponding to specific DNA fragments, thereby allowing quantitative subtraction of concurrent DNA degradation from necrotic death. Integration of the areas specifically under the peaks yields a quantitative measure of apoptosis. We provide a useful, rapid, and objective means to quantitate apoptosis, using relatively inexpensive hardware and software.
Vodovotz
Y
Y
Radiation Biology Branch, National Cancer Institute, Bethesda, Maryland 20892, USA. yoram@box-y.nih.gov
Hsing
A
A
Cook
J A
JA
Miller
R W
RW
Wink
D A
DA
Ritt
D M
DM
Mitchell
J B
JB
Danielpour
D
D
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D001345:Autoradiography; D053938:DNA Fragmentation; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008297:Male; D051381:Rats; D014407:Tumor Cells, Cultured