A simplified protocol for apoptosis assay by DNA content analysis.
Apoptotic cells possess specific morphological and biochemical markers. Various methods have been developed to detect apoptosis based on these markers. One of the most common is the fragmentation of genomic DNA. In addition to electrophoresis for the identification of the characteristic 200 base pair ladders and terminal deoxyribonucleotidyltransferase-mediated dUTP-biotin nick end-labeling, DNA content analysis is often employed. This technique is based on the fact that permeablized apoptotic cells release fragmented DNA, resulting in DNA content that is less than that in live diploid cells. Although widely used, we have found that the number of apoptotic cells detected by DNA content analysis is often lower than that detected by other methods. We have developed a simplified version of the flow cytometry-based protocol that detects a number of apoptotic cells closer to that detected by other methods, and which requires a dramatically reduced number of cells. In addition, this simplified protocol allows preparation of a large number of samples at the same time.
Wang
Ruoxiang
R
Department of Urology, Emory University School of Medicine, Atlanta, GA, USA.
Shi
Yu-Fang
YF
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D017252:CD3 Complex / Q000276:immunology; D002452:Cell Count; D002463:Cell Membrane Permeability; D002478:Cells, Cultured / Q000737:chemistry / Q000187:drug effects; D004247:DNA / Q000032:analysis*; D053938:DNA Fragmentation; D003907:Dexamethasone / Q000494:pharmacology; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D006825:Hybridomas / Q000737:chemistry / Q000187:drug effects; D007962:Leukocytes / Q000166:cytology* / Q000187:drug effects; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D012680:Sensitivity and Specificity; D019311:Staurosporine / Q000494:pharmacology; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects
D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D003907:Dexamethasone; D004247:DNA; D019311:Staurosporine