SINGLE-STEP PROCEDURE FOR LABELING DNA STRAND BREAKS WITH FLUORESCEIN-CONJUGATED 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 now 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, The cells, labeled with BrdUrd for 30 min or 1 h, could also be identified in a single-step reaction; under the present conditions, better discrimination (signal-to-noise ratio) was obtained measuring cell. fluorescence with the microscope cytometery compared to cell analysis by now cytometery. Preincubation of cells with Hoechst 33258 prior to ultraviolet (UV) illumination, to enhance the photolysis, improved detection of cells which incorporated BrdUrd. The morphology of cells subjected to SBIP was excellent, allowing visualization of distinct DNA replication points. Because, unlike the immunocytochemical methods used to detect BrdUrd incorporation, the SBIP methodology does not require DNA denaturation by heat or acid, nuclear proteins are expected to remain undenatured in situ, allowing one to study colocalization of various constituents, detected Immunocytochemically, at the DNA replication points. (C) 1995 Wiley-Liss, Inc.