BrdU-Hoechst Flow Cytometry Links the Cell Kinetic Defect of Fanconi Anemia to Oxygen Hypersensitivity

Abstract

When resting human cells are activated in media containing BrdU, thymidine residues are replaced by the halogenated analogue during semiconservative DNA replication. BrdU substitution leads to diminished fluorescence if these cells are stained with the fluorochrome Hoechst 33258. This is a principle familiar to cytogeneticists employing the differential metaphase staining technique for sister chromatid exchange (SCE) analysis (Latt 1973). By measuring the amount of Hoechst dye quenching after BrdU incorporation in a flow cytometer, one can literally count the number of times a cell has divided in the presence of BrdU (Kubbies and Rabinovitch 1983). The use of a second, BrdU-independent dye (e.g., ethidium bromide) permits the simultaneous recording of quenched and unquenched fluorescence (the latter, of course, reflecting the regular 2 c to 4 c oscillations of DNA content as cells replicate and divide). Figure 1 shows examples of such a bivariate (Hoechst fluorochrome vs. ethidium bromide) fluorescence analysis. The informational content of this type of analysis is unique since it resolves cell-cycle phase fractions throughout three successive cell cycles. By electronic framing, rotation, and projection onto the Hoechst axis, the component phases of each individual cell cycle can be separately displayed: they are then amenable to analysis by conventional curve fitting techniques (bottom panels in each figure). In the example of a 72 h phytohemagglutinin (PHA)-stimulated peripheral blood-lymphocyte culture from a healthy donor (Fig. 1 a), the cell cycle distribution of a total of 91944 cells was recorded and analyzed by curve fitting.

Supported by Deutsche Forschungsgemeinschaft grant Ho 849/2 − 1 and National Institutes of Health grant AG - 01751