Flow cytometric measurement of total DNA content and incorporated bromodeoxyuridine.

TLDR

We have developed a flow‑cytometric procedure that simultaneously measures cellular DNA content and BrdUrd incorporation. The method uses propidium iodide and a BrdUrd‑specific monoclonal antibody with a fluorescein‑labeled secondary antibody to quantify DNA and BrdUrd by flow cytometry, enabling estimation of cell‑cycle traverse rates and analysis of bone‑marrow responses to araC. The assay distinguishes G1/G2M cells with low BrdUrd fluorescence from S‑phase cells with high fluorescence and, when applied to bone‑marrow cells, reveals araC‑induced recovery kinetics and identifies sterilized cells.

Abstract

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 anti-mouse antibody was used to fluorescently label the bound anti-BrdUrd 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 BrdUrd. Bivariate DNA/BrdUrd distributions were also applied in the analysis of the response of C3H murine bone marrow cells to treatment in vivo with 1-beta-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.