Winlist 3d 8

Following incubation with drugs, 5 × 10⁵ cells were spun down and resuspended in 0.5mL HEPES solution (10mM HEPES, 150mM NaCl, 1mM MgCl₂, 5mM KCl, 1.8mM CaCl₂). Cells were incubated with 5μl FITC annexin V (Life Technologies) for 10 minutes at room temperature followed by addition of 10μl of a 50μg/ml solution of propidium iodide (Life Technologies). Flow cytometry was performed using a BD LSR II Flower Cytometer at the Flow and Image Cytometry Core facility at the Roswell Park Cancer Institute. Data was analyzed using WinList 3D 8.0 software (Verity Software House, Topsham, ME).

MDS-L cells (7.5 × 10⁵ cells/ml) were exposed to WFA (10 μM) or DMSO for 8 h. Cells treated with FCCP (50 μM) for 2 h were used as a positive control. JC-1 was added at a 1 μM final concentration to cells for the last 30 min of treatment at 37°C and fluorescence was measured by the iCyt Synergy sorter system (Sony Biotechnology Inc., San Jose, CA) with 488 and 561 nm lasers. WinList 3d 8.0 software (Verity Software House Inc., Topsham, Maine) was used for data analyses. For microscopy, cells treated with WFA (10 μM) or DMSO and stained with JC-1 as described above were mounted on poly-l Lysine (MilliporeSigma-Aldrich # P-6282) coated slides by Cytospin. Pictures were taken on the same day with a Nikon A1RSi confocal microscope (Nikon Instruments Inc, Melville, NY).

Bacterial cell counts were measured by flow cytometry using an Influx (BD Biosciences, San Jose, CA, USA) with a 200 mW 488 nm laser. Thawed samples were stained with SYBR Green I (0.5X final concentration; Molecular Probes, Inc., Eugene, OR, USA) for 15 min at room temperature in the dark [33 (link)]. 0.75 μm yellow-green and 0.5 μm green polystyrene beads (Polysciences, Inc., Warrington, PA, USA) were added as a standard. Samples were run for 2 min at 25 μl min^-1 after a 2 min pre-run, and the inline measurement was confirmed by weighing the sample before and after the run. Bacterial populations were resolved by green fluorescence (520/35 nm bandpass filter, also serving as the data collection trigger) and Forward Angle Light Scatter (FALS). Cyanobacteria were analyzed in unstained samples based on FALS (trigger) versus chlorophyll autofluorescence (692/40 nm bandpass) and phycoerythrin autofluorescence (572/27 nm bandpass, Synechococcus only). Prochlorococcus abundances from unstained samples were subtracted from bacterial abundances derived from stained samples to enumerate non-photosynthetic bacteria. Flow cytometry data were analyzed in WinList 3D 8.0 (Verity Software House, Topsham, ME, USA).