Additional Supporting Information may be found in the online version of this article.

CYTO_22066_sm_SuppFig1.tif1309KSupplemental Figure 1. Emission spectra of lipophilic dyes and cells. The fluorescence of 0.5 x 106 S. dimorphus cells/mL was measured with and without 1 µg/mL of each lipophilic dye. The fluorescence of each lipophilic dye was measured at 10 µg/mL when dye precipitation was occurring. Excitation was at 488 nm. Arrows indicate the wavelengths of the channels used during flow cytometry.
CYTO_22066_sm_SuppFig2.tif14876KSupplemental Figure 2. Dot plots showing the gating strategy for cells, debris, and precipitate. These bivariate dot plots show representative S. dimorphous data of unstained cells and cells stained with 10 µg/mL of Nile Red, BODIPY 505/515, or DiO-C18. The top panels of far red (670 – 735nm) versus infrared (750-810nm) fluorescence show the gating strategy used to isolate the precipitate and debris from the cellular events. There is clear separation of some of the precipitate from the cells, but it is not complete. The use of FSC, in the bottom panels, does not show any clear separation between the precipitate and cellular events. Blue dots represent debris, red dots represent precipitate, and green dots are cells.
CYTO_22066_sm_SuppFig3.tif525KSupplemental Figure 3. Top panels show dye fluorescence was not significantly affected by increasing the concentration of cells during staining with 1 µg/mL of lipophilic dye. There was some variation due to the need to change flow rates with different concentrations of cells. Bottom panels show the composition of the population that produced the fluorescence signal. Only at the lowest and highest cell concentrations was there a slight decrease in the percentage of cell (□) events and increase in the percentage of debris (•) precipitate (×) events. Nile Red fluorescence was measured in the yellow channel, while BODIPY fluorescence was measured in the green channel. Percentage of events data are shown as mean and standard deviation and relative fluorescence data are shown as mean and 95% confidence interval relative to unstained cells.
CYTO_22066_sm_SuppInfo.doc55KSupporting Information

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