Single-step procedure for labeling DNA strand breaks with fluorescein- or BODIPY-conjugated deoxynucleotides: detection of apoptosis and bromodeoxyuridine incorporation.
The methods of in situ labeling of DNA strand breaks have been used to identify apoptotic cells and/or DNA replicating cells. While discrimination of apoptotic cells is based on the inherent presence of numerous DNA strand breaks in their chromatin, DNA proliferating cells can be discriminated by the selective DNA strand break induction by photolysis (SBIP) methodology at the sites that contain incorporated bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd). In both instances, DNA strand breaks are labeled with biotin- or digoxygenin-conjugated deoxynucleotides, preferably in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase; fluorescein tagged avidin (streptavidin) or digoxygenin antibody is used in the second step of the reaction. In the present study, DNA strand break labeling was simplified by using directly labeled deoxynucleotides, in a single-step reaction. Cell fluorescence was measured by flow cytometry as well as by a microscope-based laser scanning multiparameter cytometer. Apoptotic cells in HL-60 cultures treated with camptothecin or in primary cultures of non-Hodgkin's lymphoma cells treated with prednisolone were easily identified utilizing BODIPY-conjugated dUTP (B-dUTP). Apoptotic cells were also recognized using fluorescein-conjugated dUTP or dATP, although the discrimination was more pronounced with B-dUTP. The single-step procedure, requiring fewer centrifugation steps, resulted in less cell loss compared to the two-step cell labeling technique.(ABSTRACT TRUNCATED AT 250 WORDS)
Li
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Cancer Research Institute, New York Medical College, Valhalla, USA.
Traganos
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Melamed
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MR
Darzynkiewicz
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Z
D017209:Apoptosis; D001483:Base Sequence; D001896:Boron Compounds; D001973:Bromodeoxyuridine / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D004261:DNA Replication; D003854:Deoxyribonucleotides; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D008969:Molecular Sequence Data; D010782:Photolysis; D014407:Tumor Cells, Cultured
D001896:Boron Compounds; D003854:Deoxyribonucleotides; D005452:Fluoresceins; D005456:Fluorescent Dyes; D004247:DNA; D001973:Bromodeoxyuridine