A Pitfall of Propidium Iodide Staining in Fluorescence-Activated Cell Sorting Cell Cycle Analysis?

Abstract

In Response:We thank Franz Buchegger and coworkers for their interest in our article. They raise a valid point about the pitfalls of propidium iodide staining in fluorescence-activated cell sorting (FACS) cell cycle analysis. Fortunately, we provide a control cell line that does not show 5-fluoro-2′-deoxyuridine (FdUrd)–induced turnover but does show a cell cycle–induced turnover of the nuclear isoform of uracil-DNA glycosylase (nUDG). In previous work, we show that nUDG from HeLa S3 cells turns over in a cell cycle–dependent manner (1). In contrast, nUDG remains stable when HeLa S3 cells are treated with FdUrd. Therefore, similar cell cycle–dependent mechanisms seem to exist for nUDG turnover in both HeLa S3 and HT29 cell lines. In contrast, different mechanisms seem to be at work in these cell lines in relation to FdUrd-induced nUDG instability. Both cell lines reveal nearly identical cell cycle patterns as a function of time post-FdUrd treatment. FACS analyses of both cell lines, as a function of FdUrd treatment, are shown in Fig. 2 of our recent publication (2). Taking our published results in their entirety tends to indicate that there is a distinct FdUrd-associated trigger that induces nUDG instability in certain cell lines (e.g., HT29) but not in others (e.g., HeLa S3).