Detection of apoptotic cells by selective precipitation of [3H]thymidine-labelled DNA.
Apoptosis is characterized by systematic fragmentation of high-molecular-weight DNA into oligonucleosome fragments. A sensitive method for detection of apoptotic cells involving [3H]thymidine-labelled DNA is presented. Cells from mid-log-phase cultures were labelled with [3H]thymidine for 15 to 18 h and then exposed to gamma irradiation to induce apoptosis. A modified Hirt method was used to separate low-molecular-weight DNA from high-molecular-weight DNA. The percentage of fragmented DNA and high-molecular-weight DNA were measured by scintillation spectrometry. This method was compared with an established flow cytometric method for detection of apoptotic cells by using propidium iodide staining of DNA. We observed a good correlation between these two methods in detecting apoptosis. Hence, expensive flow cytometric assays for detection of apoptosis in dividing cells may be replaceable by a method involving [3H]thymidine labelling of DNA and separation of low-molecular-weight DNA from high-molecular-weight DNA by precipitation.
Patki
A H
AH
Department of Medicine, Case Western Reserve University School of Medicine, University Hospitals of Cleveland, Ohio 44106, USA.
Lederman
M M
MM
D017209:Apoptosis / Q000502:physiology* / Q000528:radiation effects; D002460:Cell Line; D011232:Chemical Precipitation; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D005069:Evaluation Studies as Topic; D005434:Flow Cytometry; D005720:Gamma Rays; D006801:Humans; D008970:Molecular Weight; D011419:Propidium; D012588:Scintillation Counting; D013194:Staining and Labeling; D013936:Thymidine / Q000378:metabolism
D011419:Propidium; D004247:DNA; D013936:Thymidine