Samples were collected from the CTD-Rosette PVC Niskin bottles, then preserved with paraformaldehyde (0.5% final concentration). Preserved samples were then flash frozen in liquid nitrogen, and subsequently stored at -80\u00b0C until analysis on-shore in batches. Upon thawing, samples were stained with Hoechst 33342 DNA stain (1 ug/mL final concentration), incubated at room temperature in the dark for 1 h, then analyzed using a Beckman-Coulter Altra flow cytometer, equipped with one 488 nm 1W laser and one UV 200 mM laser for excitation. Samples (100 ul) were delivered quantitatively to the instrument using a Harvard Apparatus syringe pump, at a rate of 50 ul per minute. Signals from the DNA (450\u00b140 nm), Chlorophyll (680\u00b120 nm), Phycoerythrin (575\u00b120 nm), and the forward and side scatter detectors were used to delineate Prochlorococcus, Synechococcus, and Picophytoeukaryotes. Calibration beads (0.5 um and 1.0 um yellow-green and 0.5 um UV) were used to normalize cellular fluorescence values and assure optimal instrument settings. Raw data files (listmode) were processed in FlowJo software (Treestar Inc.).
Picophytoplankton abundances from preserved (0.5% paraformaldehyde) frozen samples run on a Beckman-Coulter Altra flow cytometer after staining with Hoechst 33342 DNA stain and excitation with 200 mW UV and 1W 488 nm laser light. Cell populations include Prochlorococcus, Synechococcus, and Picophytoeukaryotes. Samples were collected on the MV1008 cruise in the Costa Rica Dome (CRD) region of the Eastern Tropical Pacific Ocean.
Raw data, representing 100 ul of sample, was corrected for dilution with preservative, dye, and run volume, to arrive at cellular concentrations (cells/mL).