Monoclonal antibodies specific for 5-bromodeoxyuridine have been produced and applied in detecting low levels of DNA replication on a cell-by-cell basis in vitro. The immunoglobulin-producing hybridomas were derived from spleen cells of mice immunized with a conjugate of iodouridine and ovalbumin. The cells were fused with the plasmacytoma line SP2/0Ag14. The antibodies produced are highly specific for bromodeoxyuridine and iododeoxyuridine and do not cross-react with thymidine. DNA synthesis in cultured cells exposed to bromodeoxyuridine for as short a time as 6 minutes can be detected easily and rapidly by an immunofluorescent staining method and quantitated by flow cytometry.

We have developed a procedure for simultaneous flow cytometric measurement of cellular DNA content and amount of BrdUrd incorporated into cellular DNA. Propidium iodide was used as a fluorescent probe for total cellular DNA and a monoclonal antibody against BrdUrd was used as a probe for BrdUrd incorporated into DNA. Fluorescein-labeled goat antimouse antibody was used to fluorescently label the bound antiBrdUrd probe. Bivariate DNA/BrdUrd distributions measured for Chinese hamster ovary cells labeled for 30 min with BrdUrd clearly show the G1-and G2M-phase cells to have low BrdUrd-linked fluorescence and the S-phase cells to have high BrdUrd-linked fluorescence. Cell cycle.traverse rates were estimated for Chinese hamster ovary cells from bivariate distributions measured-for samples taken periodically after pulse labeling with BrdUrdw Bivariate DNA/BrdUrd distributions were also applied in the analysis of the response of C3H murine bone marrow cells to treatment in vivo with l-I3-D-arabinofuranosylcytosine (araC). Bivariate distributions were measured for bone marrow cells taken from mice that were pulse labeled with BrdUrd at various times after treatment with araC. The resulting DNA/BrdUrd sequences show the kinetics of recovery from araC and allow discrimination of the araC sterilized cells.A broad range of biological and biomedical investigations depends on the ability to distinguish DNA synthesizing cells. Oncologists, for example, have devoted substantial effort to establishing correlations between the frequency of DNA synthesizing human tumor cells and the treatment prognosis (1). Effort has also been devoted to improvement of anticancer therapy with S-phase specific agents by treating when the experimentally determined frequency of tumor cells in S phase is maximal (2). In these studies, S-phase cells are usually assumed to be those that appear labeled in autoradiographs prepared immediately after pulse labeling with [3H]dThd or those with S-phase DNA content in DNA distributions measured flow cytometrically. Cytokineticists have relied heavily on measurements of the frequency of DNA synthesizing cells to determine the cell cycle traverse characteristics of normal and malignant cells. The classical "fraction of labeled mitosis" procedure (3), for example, depends on assessment of the frequency of mitotic cells that appear radioactively labeled in autoradiographs of samples taken periodically after labeling with [3H] We now report the development of a powerful, yet simple, flow cytometric procedure that promises to decrease or remove many of the limitations inherent in earlier methods for study of cell proliferation. Our procedure is based on the simultaneous flow cytometric measurement of cellular DNA content and amount of incorporated BrdUrd. For these measurements, propidium iodide (PrdI) is used as the probe for total DNA content and a monoclonal antibody against BrdUrd (8) Fig. 2 Upper. Ten thousand cells were sorted from each region directly into liquid scintillation vials. The radioactivi...

No abstract

The flow cytometric measurement of DNA distributions of cells has many applications in biomedical research. Phase fractions estimated (calculated) from such distributions are used to study the growth characteristics of various types of cells, particularly when the cells have been exposed to perturbing agents such as chemotherapeutic drugs. For more than 10 years many methods for resolving DNA

No abstract