Samples were taken using a rosette of Niskin bottles, fixed with freshly titrated paraformaldehyde (pH 7.4–8.1, 0.1% final concentration), held in the dark for 10 min, frozen in liquid nitrogen, and stored in a -80 deg C freezer (CH0409 samples) or in liquid nitrogen (CH0510 samples) until analysis. Preserved samples were analyzed by dual beam flow cytometry on a modified Coulter-EPICS 753 flow cytometer (Binder et al. 1996). Samples were chosen in random order, defrosted in a 30°C water bath (just long enough to melt, ~5 min), and stained with the DNA-specific stain Hoechst 33342 (0.5 ug mL-1 final concentration) (Invitrogen, Carlsbad, California) for a minimum of 20 min in the dark. Prior to analysis, polystyrene fluorescent beads (0.5 um and 1.0 um diameter Flow CheckVR ; Polysciences, Washington, Pennsylvania), were added to each sample, and used to normalize cellular light scatter and red (chlorophyll-derived) fluorescence. Samples were run at an infusion rate of 10 uL min-1 for 10–50 min, depending on cell abundance within the sample.

Mean red fluorescence and forward angle light scatter for each cell type in each sample are linearized and normalized to 1.0 um and 0.5 um diameter beads (see above), respectively. Thus the measurements are relative, and are only meaningful for comparisons of cellular properties within this data set.