DNA Content Analysis and Drug Interactions Figure 5. A novel interaction between gemfibrozil and fluoxetine. A, Fluoxetine strongly inhibits yeast cell proliferation, but it is suppressed by gemfibrozil. We added to freshly reseeded wild type haploid yeast (BY4741) cells DMSO, fluoxetine and gemfibrozil at the binary combinations and concentrations shown. We then monitored cell proliferation hourly, for 8 h (see Materials and Methods). The specific growth rate constant (k) for each combination is shown. The errors associated with these measurements are shown in Table S2. B, DMSO, fluoxetine and gemfibrozil were added to dividing cells at 200 mM in binary combinations, sequentially, in the order shown. Cell proliferation was monitored for 6 h as in a, with the specific growth rate constant (k) for each combination shown. Data from one representative experiment is shown. Suppressive effects of gemfibrozil on fluoxetine arising from order of addition were assessed by calculating growth rate constant (k) folds for gemfibrozil treatment over DMSO control for all experiments, initial treatment with gemfibrozil yielding a fold of 2.51 +/2 0.25, versus final treatment, 0.71 +/2 0.21, P-value = 0.000146. doi:10.1371/journal.pone.0036503.g005 0.25 mg/ml RNaseA, and 1 mM SYTOX Green (Molecular Probes). Samples were stored at 4uC overnight in opaque containers. Cell suspensions were sonicated briefly at the fixing and staining steps and immediately before flow cytometry. Flow cytometry data acquisition, deposition and analysis Stained cells were analyzed on a FACSCalibur (Becton Dickinson Immunocytometry Systems) flow cytometer, using CellQuest (version 3.3; Becton Dickinson Immunocytometry Systems) acquisition software. Sytox Green fluorescence was collected through a 515/30-nm bandpass filter, and list mode data were acquired for 10,000 cells defined by a dot plot of FSC versus SSC. Prior to each experiment, standard beads (Cyto-Cal Multifluor Intensity Beads, Thermo Scientific) were used to calibrate the flow cytometer, and photomultiplier tube voltages were adjusted to place the highest intensity bead in the same channel (+/2 3). FACS files were archived at Cytobank. Automated quantification of the DNA content histograms was done with FlowJo 7.5 software. To exclude particulate non-yeast events, which had both very low forward scatter (FSC) and low fluorescence (FL2-A), asymmetrical gates were fitted with the autogating tool. Gates were centered near FSC ,100 and FL2-A PLoS ONE | www.plosone.org 7 May 2012 | Volume 7 | Issue 5 | e36503