Detection of apoptosis and DNA replication by differential labeling of DNA strand breaks with fluorochromes of different color.

摘要: Abstract Selective DNAstrandbreakinduction byphotolysis (SBIP) at sites that contain incorporated halogenated nucleotides has been recently proposed as a means of analyzing DNA replication and detecting proliferating cells. The presence numerousin situDNA strand breaks is also an inherent feature apoptotic aim the present study was to differentially label in cells vs photolysis-induced BrdUrd incorporating This would allow one, by multicolor staining, identify these respective same sample preparation. Toward this end, exponentially growing HL-60 were pulse labeled with then subjected hyperthermia or treated topoisomerase I inhibitor camptothecin induce apoptosis. first directly fluorochrome-conjugated dUTP dCTP, followed dideoxynucleotide (to terminate chain elongation), reaction catalyzed exogenous terminal deoxynucleotidyl transferase. subsequently exposed UV light photolyze containing BrdUrd. were, turn, digoxygenin- biotin-conjugated digoxygenin antibody avidin, respectively, conjugated fluorochrome another color. Alternatively, BrdUTP which detected FITC-conjugated anti-BrdUrd MoAb. Following counterstaining cellular third color it possible cells, BrdUrd, having no breaks. Cell fluorescence measured either flow cytometry microscope-based laser scanning cytometer. SBIP approach offers possibility colocalization immunocytochemically detectable cell constituents points microscopy. Using nuclear antigen revealed MCF-7 breast carcinoma