Cell cycle analysis with EdU using flow cytometry. Formaldehyde fixed (A-D and G) or unfixed (E, F) nuclei isolated from EdU-treated and untreated rice (A, B, E, F), alfalfa (C, D) and Arabidopsis (G) cultures were analyzed by flow cytometry. Uniparametric DNA histograms of propidium iodide intensity (FL2-A) are shown at upper panels. Lower panels show biparametric dot-plot analysis of Alexa Fluor 488-EdU intensity (FL1-H, logarithmic scale) versus propidium iodide intensity (FL2-H). Braces in biparametric plots indicate the total EdU-labeled population or background green fluorescence above EdU threshold (see Methods). Inset photos show fluorescence images of isolated nuclei counterstained with DAPI (red) following EdU assay with Alexa Fluor 488 azide (green). Scalebars are 5 μm. An example of quadrant thresholds is shown for biparametric plot of Figure 5B. The ratios of nuclei in each quadrant are displayed as percentage on the lower right corners of each biparametric plot. (G) Ratio of indicated biparametric fractions (quadrants Q1-Q4) after EdU (15' and 30') and DMSO treatment of Arabidopsis suspension cultures.